AU778759B2 - IL-1 related polypeptides - Google Patents

Description

IL-1 RELATED POLYPEPTIDES FIELD OF THE INVENTION The present invention relates generally to the identification and isolation of novel DNAs having homology to interleukin-1 (IL-1) or interleukin-1 receptor antagonist (IL-1Ra) polypeptides, and to the recombinant production of novel polypeptides, designated herein as interleukin-1-like polypeptides ("IL-llp").

BACKGROUND OF THE INVENTION Interleukin-1 refers to two proteins (IL-la and IL- 1) which play a key role early in the inflammatory response (for a review, see Dinarello, Blood, 87: 2095-2147 (1996) and references therein), both proteins are made as intracellular precursor proteins which are cleaved upon secretion to yield mature carboxy-terminal 17 kDa fragments which are biologically active. In the case of IL-1p, this cleavage involves an intracellular cysteine protease, known as ICE, which is required to release the active fragment from the inactive precursor. The precursor of IL-la is active.

These two proteins act by binding to cell surface receptors found on almost all cell types and triggering a range of responses either alone or in concert with other secreted factors. These include effects on proliferation fibroblasts, T cells), apoptosis A375 melanoma cells), cytokine induction of TNF, IL-1, IL-8), receptor activation Eselectin), eicosanoid production PGE2) and the secretion of degradative enzymes (e.g.

collagenase). To achieve these effects, IL-1 activates transcription factors such as NF-KB and AP-1. Several of the activities of IL-1 action on target cells are believed to be mediated through activation of kinase cascades that have also been associated with cellular stresses, such as the stress-activated MAP kinase JNK/SAPK and p38.

A third member of the IL-1 family was subsequently discovered which acts as a :25 natural antagonist of IL-la and IL-lP by binding to the IL-1 receptor but not transducing an intracellular signal or a biological response. The protein is called IL-1Ra (for IL-I receptor 0 antagonist) or IRAP (for IL-1 receptor antagonist protein). At least three alternatively spliced forms of IL-1Ra exist: one encodes a secreted protein, also known as secretory IL-1Ra ("sIL- 1Ra") (described in Eisenberg et al., Nature, 343: 341-346 (1990)), and the other two encode 30 intracellular proteins. IL-la, IL-1P and IL-1Ra exhibit approximately 25-30% sequence identity with each other and share a similar three dimensional structure consisting of twelve P-strands folded into a P-barrel, with an internal thrice repeated structural motif.

•There are three known IL-1 receptor subunits. The active receptor complex consists of the type I receptor and IL-1 accessory protein (IL-1RAcP). The type I receptor is 35 responsible for binding of the IL-la, IL-10 and IL-1Ra ligands, and is able to do so in the absence of the IL-1RAcP. However, signal transduction requires the interaction of IL-la or IL-1p with the IL-1RAcP. IL-1Ra does not interact with the IL-1RAcP and hence cannot induce signal transduction. A third receptor subunit, the type II receptor, binds IL-la and IL-1l but cannot transduce signal due to its lack of an intracellular domain. Instead, the H:Junita\Keep\pwcrn\S935.4 doc 8/1004 type II receptor either acts as a decoy in its membrane-bound form, or as an IL-1 antagonist in a processed, secreted form, and hence inhibits IL-1 activity. The type II receptor weakly binds to IL-1Ra.

Many studies using IL-1Ra, soluble IL-1R derived from the extracellular domain of the type I IL-1 receptor, antibodies to IL-la or IL-1p, and transgenic knockout mice for these genes have shown that IL-1 plays a role in a number of pathophysiologies (for a review, see Dinarello, Blood, 87: 2095-2147 (1996)). For example, IL-1Ra has been shown to be effective in animal models of septic shock, rheumatoid arthritis, graft-versus-host disease (GVHD), stroke, cardiac ischemia, psoriasis, inflammatory bowel disease, and asthma. In addition, IL-lRa has demonstrated efficacy in clinical trials for rheumatoid arthritis and GVHD, and is also in clinical trials for inflammatory bowel disease, asthma and psoriasis.

More recently, interleukin-18 (IL-18) was placed in the IL-1 family (for a review, see Dinarello et al, J. Leukocvte Biol., 63: 658-664 (1998)). IL-18 shares the P-pleated, barrellike form of IL-la and IL-1p. In addition, IL-18 is the natural ligand for the IL-1 receptor family member formerly known as IL-1R-related protein (IL-1Rrp) (now known as the IL-18 receptor (IL-18R)). IL-18 has been shown to initiate the inflammatory cytokine cascade in a mixed population of peripheral blood mononuclear cells (PBMCs) by triggering the constitutive IL-18 receptors on lymphocytes and NK cells, inducing TNF production in the activated cells. TNF, in turn, stimulates IL-1 and IL-8 production in CD14+ cells. Because of its ability to induce TNF, IL-1, and both C-C and C-X-C chemokines, and because IL-18 induces Fas ligand as well as nuclear translocation of nuclear factor KB (NF-KB), IL-18 ranks with other pro-inflammatory cytokines as a likely contributor to systemic and local I inflammation.

All references, including any patents or patent applications, cited in this specification are 25 hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to Schallenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in 30 Australia or in any other country.

SUMMARY OF THE INVENTION A family of cDNA clones (DNA85066, DNA96786, DNA94618, DNA102043, DNA114876, DNA102044, and DNA92929 has been identified, which encode novel polypeptides having homology to interleukin-1. The novel polypeptides and variants thereof 35 are collectively designated in the present application as "interleukin-1-like polypeptides" or "IH-11p", as further defined herein. Accordingly, one aspect of the invention is an isolated Lllp polypeptide.

In another embodiment, the invention provides an isolated nucleic acid molecule encoding an IL-llp polypeptide.

H:Uuanita\Keep\pazcnt\25935.4 do 8/10/04 In another embodiment, the invention provides a method for producing an IL-lip, comprising culturing a host cell comprising a heterologous nucleic acid sequence encoding

S

0 S S -2a- Hs\Juanita\Keep\patelt\25935.4 .doc 8/10/04 an IL-llp polypeptide, under conditions wherein the IL-llp polypeptide is expressed, and recovering the IL-llp polypeptide from the host cell.

In another embodiment, the invention provides an anti-lL-llp antibody.

In another embodiment, the invention provides chimeric molecules comprising an ILlip polypeptide fused to a heterologous polypeptide or amino acid sequence. An example of such a chimeric molecule comprises an IL-11p polypeptide fused to an epitope tag sequence or a Fc region of an immunoglobulin.

In another embodiment, the invention provides an antibody which specifically binds to an IL-llp polypeptide. Optionally, the antibody is a monoclonal antibody.

In yet another embodiment, the invention concerns agonists and antagonists of a native IL-llp polypeptide. In a particular embodiment, the agonist or antagonist is an anti- IL-llp antibody.

In a further embodiment, the invention concerns a method of identifying agonists or antagonists of a native IL-llp polypeptide, by contacting the native IL-llp polypeptide with a candidate molecule and monitoring a biological activity mediated by said polypeptide.

In a still further embodiment, the invention concerns a composition comprising an ILllp polypeptide, or an agonist or antagonist as hereinabove defined, in combination with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a nucleotide sequence (SEQ ID NO:1) and derived amino acid sequences (SEQ ID NOS:2-3) related to a native sequence hIL-1Ral. The nucleotide sequence (SEQ ID NO:1) contains an intron believed to extend from nucleotide positions 181 to 432, with a splice donor site at nucleotide positions 181 to 186 and splice acceptor site at nucleotide positions 430 to 432. The amino acid sequences (SEQ ID NOS:2 and 3) are 25 derived from the exonic sequences that are believed to make up the processed (intron-free) coding sequence.

Figure 2 shows the nucleotide sequence (SEQ ID NO:4) and derived amino acid sequence (SEQ ID NO:5) of a native sequence hIL-IRal polypeptide fused at its N-terminus to a heterologous signal peptide (amino acid positions 1-15), flag peptide affinity handle (amino acid positions 16-23) and peptide linker (amino acid positions 24-36).

Figure 3 shows the nucleotide sequence (SEQ ID NO:6) and derived amino acid sequence (SEQ ID NO:7) of a native sequence hIL-1Ral polypeptide. The nucleotide sequence (SEQ ID NO:6) and derived amino acid sequence (SEQ ID NO:7) are believed to represent the processed (intron-free) form and intact hIL-1Ral polypeptide, respectively, of 35 the nucleotide sequence (SEQ ID NO:1) and amino acid sequences (SEQ ID NOS:2-3) of S: Figure 1. The start and stop codons in the coding sequence are located at nucleotide positions 103-105 and 682-684, respectively. The putative signal sequence extends from amino acid positions 1 to 14. A putative cAMP- and cGMP-dependent protein kinase phosphorylation site is located at amino acid positions 33-36. Putative N-myristoylation sites are located at amino acid positions 50-55 and 87-92.

Figure 4 shows the nucleotide sequence (SEQ ID NO:8) of EST AIO14548.

-3- Figure 5 shows the nucleotide sequence (SEQ ID NO:9) and derived amino acid sequence (SEQ ID NO:10) of a native sequence hIL-1Ra2 polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 96-98 and 498-500, respectively. The putative signal sequence extends from amino acid positions 1-26.

Figure 6 shows the nucleotide sequence (SEQ ID NO: 11) of EST 1433156.

Figure 7 shows the nucleotide sequence (SEQ ID NO:12) and derived amino acid sequence (SEQ ID NO:13) of a native sequence hIL-lRa3 polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 1-3 and 466-468, respectively. The putative signal sequence extends from amino acid positions 1-33. Putative N-myristoylation sites are located at amino acid positions 29-34, 30-35, 60-65, 63-68, 73-78, 91-96 and 106-111. An interleukin-1-like sequence is located at amino acid positions 111- 131.

Figure 8 shows the nucleotide sequence (SEQ ID NO: 14) of EST 5120028.

Figure 9 shows the nucleotide sequence (SEQ ID NO:15) and derived amino acid sequence (SEQ ID NO:16) of a native sequence mIL-1Ra3 polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 145-147 and 610-612, respectively. The putative signal sequence extends from amino acid positions 1-33. Putative N-myristoylation sites are located at amino acid positions 29-34, 60-65, 63-68, 91-96 and 106-111. An interleukin-1-like sequence is located at amino acid positions 111-131.

Figure 10 shows the nucleotide sequence (SEQ ID NO: 17) of EST W08205.

Figure 11 is an autoradiograph of Northern blots depicting expression of hIL-1Ra3 mRNA in placental tissue and expression of mIL-1Ra3 mRNA in day-17 mouse embryo tissue.

Figure 12 is an amino acid sequence alignment of native sequence hIL-lRalL (SEQ ID 25 NO:19), hIL-1RalV (SEQ ID NO:25), hlL-1RalS (SEQ ID NO:21), hIL-lRa2 (SEQ ID hIL-lRa3 (SEQ ID NO:13) and mIL-lRa3 (SEQ ID NO:16) polypeptides with secretory hIL-IRa (also referred to as "sIL-IRa" and "hIL-1Ra") (SEQ ID NO:26), hIL-1Ra3 (SEQ ID NO:27) and i TANGO-77 (SEQ ID NO:28).

Figure 13A is a Western blot depicting the interleukin-18 receptor (IL-18R) binding activity of hIL-1Ral. In the top panel (depicting a protein band at approximately 22 kD), a conditioned medium containing FLAGhIL-1Ral and FLAGIL-1R-ECD-Fc (shown in the left lane) and a conditioned medium containing FLAGhIL-1Ral and FLAGIL-18R-ECD-Fc (shown in the right lane) were each immunoprecipitated with protein G-sepharose, and the resulting precipitates were resolved by gel electrophoresis and Western blotting with anti-FLAG S: 35 monoclonal antibody. In the middle and bottom panels (depicting protein bands at S* approximately 22 kD and 85 kD), a second aliquot from the FLAGhIL-lRal and FLAGIL-1R- S ECD-Fc conditioned medium used in the top panel (shown in the left lane) and a second aliquot from the FLAGhIL-IRal and FLAGIL-18R-ECD-Fc conditioned medium used in the top panel (shown in the right lane) were each immunoprecipitated with anti-FLAG monoclonal antibody, and the resulting precipitates were resolved by gel electrophoresis and Western blotting with anti-FLAG monoclonal antibody.

-4- Figure 13B is a Western blot depicting the IL-1R binding activity of hIL-1Ra3. In the top panel (depicting a protein band at approximately 20 kD), a conditioned medium containing hIL-lRa3-FLAG and FLAGDR6-Fc (shown in the left lane), a conditioned medium containing hIL-1Ra3-FLAG and FLAGIL-lR-ECD-Fc (shown in the middle lane), and conditioned medium containing hIL-1Ra3-FLAG and FLAGIL-18R-ECD-Fc (shown in the right lane) were each immunoprecipitated with protein G sepharose, and the resulting precipitates were resolved by gel electrophoresis and Western blotting with anti-FLAG monoclonal antibody. In the middle and bottom panels (depicting protein bands at approximately 20 kD and 85 kD), a second aliquot from the hIL-IRa3-FLAG and FLAGDR6- Fc conditioned medium used in the top panel (shown in the left lane), a second aliquot from the hIL-1Ra3-FLAG and FLAGIL-lR-ECD-Fc conditioned medium used in the top panel (shown in the middle lane) and a second aliquot from the hIL-1Ra3-FLAG and FLAGIL-18R- ECD-Fc conditioned medium used in the top panel (shown in the right lane) were each immunoprecipitated with anti-FLAG monoclonal antibody, and the resulting precipitates were resolved by gel electrophoresis and Western blotting with anti-FLAG monoclonal antibody.

Figure 14 is a Western blot depicting the interleukin-1 receptor (IL-1R) binding activity of mIL-1Ra3. In the top panel (depicting a protein band at approximately 21 kD) and the bottom panel (depicting protein bands at approximately 85 kD) the FLAGIL-1R-ECD-Fc in conditioned medium (shown in the left lane) and the FLAGIL-18R-ECD-Fc in conditioned medium (shown in the right lane) were immobilized with protein G-agarose, the resulting solid phase was contacted with conditioned medium containing FLAGmIL-1Ra3, and the resulting bound complexes were resolved by gel electrophoresis and Western blotting with anti-FLAG monoclonal antibody.

25 Figure 15 shows the nucleotide sequence (SEQ ID NO:18) and derived amino acid S sequence (SEQ ID NO:19) of a native sequence hIL-1RalL polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 4-6 and 625-627, 9respectively. The putative signal sequence extends from amino acid positions 1 to 34. A putative cAMP- and cGMP-dependent protein kinase phosphorylation site is located at amino acid positions 47-50. Putative N-myristoylation sites are located at amino acid positions 64- 69 and 101-106.

Figure 16 shows the nucleotide sequence (SEQ ID NO:20) and derived amino acid sequence (SEQ ID NO:21) of a native sequence hIL-1RalS polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 4-6 and 505-507, 35 respectively. A putative signal sequence extends from amino acid positions 1 to 46. A putative N-myristoylation site is located at amino acid positions 61-66.

:Figure 17 shows the single stranded nucleotide sequence (SEQ ID NO:23) of EST AI343258 (lower strand) along with its complementary nucleotide sequence (SEQ ID NO:22) (upper strand).

Figure 18 is an amino acid sequence alignment of native sequence hIL-1Ral (SEQ ID NO:3), hIL-1RalL (SEQ ID NO:19), hIL-1RalV (SEQ ID NO:25) and hIL-lRalS (SEQ ID NO:21) polypeptides.

Figure 19 shows the nucleotide sequence (SEQ ID NO:24) and derived amino acid sequence (SEQ ID NO:25) of a native sequence hIL-1RalV polypeptide. The start and stop codons in the coding sequence are located at nucleotide positions 73-75 and 727-729, respectively. An alternate start codon is located at nucleotide positions 106-108. A putative signal sequence extends from amino acid positions 1 to 48.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I. Definitions: In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The terms "interleukin-1-like polypeptide", "interleukin-1-like protein", "IL-llp", "ILllp polypeptide", and "IL-llp protein" encompass any native sequence IL-llp, and further encompass IL-llp variants (which are further defined herein). The IL-llp may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant and/or synthetic methods.

A "native sequence IL-llp" comprises a polypeptide having the same amino acid sequence as a native sequence hIL-1Ral, hIL-1RalL, hIL-1RalV, hIL-1RalS, hIL-1Ra2, hIL- 1Ra3, or mIL-1Ra3, (which are further defined herein). Such native sequence IL-llp can be isolated from nature or can be produced by recombinant and/or synthetic means. The term S 25 "native sequence IL-11p" specifically encompasses naturally-occurring truncated or secreted forms a processed, mature sequence) and naturally-occurring allelic variants of the ILlp.

The terms "naturally-occurring amino acid sequence" and "native amino acid :i sequence" mean any amino acid sequence found in a polypeptide existing in nature, i.e.

30 present in a naturally-occurring polypeptide.

The terms "non-naturally-occurring amino acid sequence" and "non-native amino acid sequence" mean any amino acid sequence not found in a polypeptide existing in nature, i.e. not present in a naturally-occurring polypeptide.

"IL-llp variant" is defined as any polypeptide that comprises a variant of hIL-1Ral, 35 hIL-1RalL, hIL-1RalV, hIL-1RalS, hIL-1Ra2, hIL-1Ra3, or mIL-1Ra3 (which are further defined herein Human interleukin-1 receptor antagonist analog 1 ("hIL-1Ral"), hIL-1Ral polypeptide, and hIL-1Ral protein are defined as any native sequence hIL-1Ral or variant hIL-1Ral.

A "native sequence hIL-1Ral" means a polypeptide comprising a naturally-occurring amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 37 to at or about 63 of Figure 2 (SEQ ID NO:5); the amino acid sequence of amino acid residues from at or about 37 to at or about 203 of Figure 2 (SEQ ID NO:5); the amino acid sequence of amino acid residues from at or about 15 to about 53 of Figure 3 (SEQ ID NO:7); the amino acid sequence of amino acid residues from at or about 15 to at or about 193 of Figure 3 (SEQ ID NO:7); and the amino acid sequence of any naturally-occurring truncated or secreted form or any naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence of or or or In one embodiment of the invention, the native sequence hIL-1Ral comprises amino acids from at or about 37 to at or about 203 of Figure 2 (SEQ ID NO:5) or amino acids from at or about to at or about 193 of Figure 3 (SEQ ID NO:7).

"hIL-1Ral variant" is defined as any hIL-1Ral N-terminal variant or hIL-1Ral full sequence variant (which are further defined herein).

"hIL-1Ral N-terminal variant" means any hIL-1Ral other than a native sequence hIL- 1Ral, which variant is an active hIL-1Ral, as defined below, having at least about amino acid sequence identity with an amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 37 to at or about 63 of Figure 2 (SEQ ID NO:5); and the amino acid sequence of amino acid residues from at or about 15 to at or about 53 of Figure 3 (SEQ ID NO:7). Such hIL-1Ral N-terminal variants include, for instance, hIL-1Ral polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 37 to at or about 63 of Figure 2 (SEQ ID NO:5) or in the sequence of amino acid residues from at or about 15 to at or about 53 of Figure 3 (SEQ ID NO:7). Ordinarily, an hIL-1Ral N-terminal variant will have at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about amino acid sequence identity, or at least about 95% amino acid sequence identity with an amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 37 to at or about 63 of Figure 2 (SEQ ID NO:5); and (2) 30 the amino acid sequence of amino acid residues from at or about 15 to at or about 53 of S* Figure 3 (SEQ ID NO:7).

"hIL-1Ral full sequence variant" means any hIL-1Ral other than a native sequence hIL-1Ral, which variant retains at least one biologic activity of a native sequence hIL-1Ral, S: such as the ability to bind IL-18R, and which variant has at least about 80% amino acid 35 sequence identity, or at least about 85% amino acid sequence identity, or at least about amino acid sequence identity, or at least about 95% amino acid sequence identity with an amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 37 to at or about 203 of Figure 2 (SEQ ID NO:5); and the amino acid sequence of amino acid residues from at or about 15 to at or about 193 of Figure 3 (SEQ ID NO:7). Such hIL-1Ral full sequence variants include, for instance, hIL- IRal polypeptides wherein one or more amino acid residues are added, or deleted, internally -7or at the N- or C-terminus, in the sequence of amino acid residues from at or about 37 to at or about 203 of Figure 2 (SEQ ID NO:5) or in the sequence of amino acid residues from at or about 15 to at or about 193 of Figure 3 (SEQ ID NO:7).

Human interleukin-1 receptor antagonist analog 1 long ("hIL-1RalL"), hIL-1RalL polypeptide, and hIL-1RalL protein are defined as any native sequence hIL-1RalL or hIL- 1Ra 1L variant (which are further defined herein).

A "native sequence hIL-1Ra 1 L" means a polypeptide comprising a naturally-occurring amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 26 to at or about 44 of Figure 15 (SEQ ID NO:19); (2) the amino acid sequence of amino acid residues from at or about 26 to at or about 207 of Figure 15 (SEQ ID NO:19); and the amino acid sequence of any naturally-occurring truncated or secreted form or any naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence of or In one embodiment of the invention, the native sequence hIL-1RalL comprises amino acids from at or about 26 to at or about 207 of Figure 15 (SEQ ID NO:19).

"hIL-RalL variant" is defined as any hIL-1RalL N-terminal variant or hIL-1RalL full sequence variant or hIL-1RalL fusion variant (which are further defined herein).

"hIL-1RalL N-terminal variant" means any hIL-1RalL other than a native sequence hIL-1RalL, which variant is an active hIL-1RalL, as defined below, having at least about 80% amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 26 to at or about 44 of Figure 15 (SEQ ID NO:19). Such hIL-1RalL N-terminal variants include, for instance, hIL-1RalL polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 26 to at or about 44 of Figure 15 (SEQ ID NO:19).

Ordinarily, an hIL-1RalL N-terminal variant will have at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about amino acid sequence identity, or at least about 95% amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 26 to at or about 44 of Figure 15 (SEQ ID NO:19).

30 "hIL-1RalL full sequence variant" means any hIL-1RalL other than a native sequence hIL-1RalL, which variant retains at least one biologic activity of a native sequence hIL- 1RalL, such as the ability to bind IL-18R, and which variant has at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% amino acid sequence identity with S: 35 the amino acid sequence of amino acid residues from at or about 26 to at or about 207 of Figure 15 (SEQ ID NO:19). Such hIL-1RalL full sequence variants include, for instance, hIL- S 1RalL polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 26 to at or about 207 of Figure 15 (SEQ ID NO: 19).

"hIL-1RalL fusion variant" means a chimeric hIL-1RalL consisting of a native sequence hIL-1RalL fused at its N- or C-terminus to a heterologous amino acid or amino -8acid sequence. In one embodiment, the hIL-1RalL fusion variant polypeptide consists of a native sequence of hIL-1RalL fused at its N-terminus or C-terminus to a heterologous amino acid or amino acid sequence, wherein the heterologous amino acid or amino acid sequence is heterologous to the native sequence; i.e. the resulting chimeric sequence is non-naturally occurring. In another embodiment, the hIL-1RalL fusion variant consists of the amino acid sequence of amino acids from at or about 26 to at or about 207, inclusive of Figure 15 (SEQ ID NO:19), or the amino acid sequence of amino acid residues from at or about 1 to at or about 207, inclusive of Figure 15 (SEQ ID NO:19), fused at its N-terminus or C-terminus to a heterologous amino acid or amino acid sequence to form a non-naturally occurring fusion protein. Such hIL-1RalL fusion variants include, for instance, hIL-1RalL polypeptides wherein a heterologous secretion leader sequence is fused to the N-terminus of the sequence of amino acid residues from at or about 26 to at or about 207 of Figure 15 (SEQ ID NO: 19), or amino acid residues from at or about 1 to at or about 207 of Figure 15 (SEQ ID NO: 19).

Human interleukin-1 receptor antagonist analog 1 long allelic variant ("hIL-1RalV"), hIL-1RalV polypeptide, and hIL-1RalV protein are defined as any native sequence hIL- 1RalV or hIL-1RalV variant (which are further defined herein).

A "native sequence hIL-1RalV" means a polypeptide comprising a naturally-occurring amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 46 to at or about 55 of Figure 19 (SEQ ID NO:25); (2) the amino acid sequence of amino acid residues from at or about 46 to at or about 218 of Figure 19 (SEQ ID NO:25); the amino acid sequence of amino acid residues from at or about 37 to at or about 218 of Figure 19 (SEQ ID NO:25); the amino acid sequence of amino acid residues from at or about 12 to at or about 218 of Figure 19 (SEQ ID NO:25); and the amino acid sequence of any naturally-occurring truncated or secreted form or any naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence of (1) or or or In one embodiment of the invention, the native sequence hIL-1RalV comprises amino acids from at or about 46 to at or about 218 of Figure 19 (SEQ ID or amino acids from at or about 37 to at or about 218 of Figure 19 (SEQ ID NO:25), or amino acids from at or about 12 to at or about 218 of Figure 19 (SEQ ID NO:25), or amino acids 30 from at or about 1 to at or about 218 of Figure 19 (SEQ ID "hIL-1RalV variant" is defined as any hIL-1RalV N-terminal variant or hIL-1RalV full sequence variant or hIL-1RalV fusion variant (which are further defined herein).

.:"hIL-1RalV N-terminal variant" is defined as any hIL-1RalV other than a native sequence hIL-1RalV, which variant is an active hIL-1RalV, as defined below, having at least 35 about 80% amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 46 to at or about 89 of Figure 19 (SEQ ID NO:25). Such hIL-1RalV N-terminal variants include, for instance, hIL-IRalV polypeptides wherein one or more amino acid residues are added, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 46 to at or about 89 of Figure 19 (SEQ ID Ordinarily, an hIL-1RalV N-terminal variant will have at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about amino acid sequence identity, or at least about 95% amino acid sequence identity with the sequence of amino acid residues from at or about 46 to at or about 89 of Figure 19 (SEQ ID "hIL-1RalV full sequence variant" means any hIL-1RalV other than a native sequence hIL-1RalV, which variant retains at least one biologic activity of a native sequence hIL-1RalV, such as the ability to bind IL-18R, and which variant has at least about amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% amino acid sequence identity with the sequence of amino acid residues from at or about 46 to at or about 218 of Figure 19 (SEQ ID "hIL-1RalV fusion variant" means a chimeric hIL-1RalV consisting of a native sequence hIL-1RalV fused at its N- or C-terminus to a heterologous amino acid or amino acid sequence. In one embodiment, the hIL-1RalV fusion variant polypeptide consists of a native sequence of hIL-1RalV fused at its N-terminus or C-terminus to a heterologous amino acid or amino acid sequence, wherein the heterologous amino acid or amino acid sequence is heterologous to the native sequence, i.e. the resulting chimeric sequence is non-naturally occurring. In another embodiment, the hIL-1RalV fusion variant consists of the amino acid sequence of amino acids from at or about 46 to at or about 218 of Figure 19 (SEQ ID or the amino acid sequence of amino acids from at or about 37 to at or about 218 of Figure 19 (SEQ ID NO:25), or the amino acid sequence of amino acids from at or about 12 to at or about 218 of Figure 19 (SEQ ID NO:25), or the amino acid sequence of amino acids from at or about 1 to at or about 218 of Figure 19 (SEQ ID NO:25), fused at its N-terminus or Cterminus to a heterologous amino acid sequence to form a non-naturally occurring fusion protein. Such hIL-1RalV fusion variants include, for instance, hIL-1RalV polypeptides wherein a heterologous secretion leader sequence is fused to the N-terminus of the sequence of amino acid residues from at or about 46 to at or about 218 of Figure 19 (SEQ ID Samino acid residues from at or about to at or about 218 of Figure 19 (SEQ ID or amino acid residues from at or about 37 to at or about 218 of Figure 19 (SEQ ID S or amino acid residues from at or about 1 to at or about 218 of Figure 19 (SEQ ID 30 Human interleukin-1 receptor antagonist analog 1 short ("hIL-1RalS"), hIL-1RalS polypeptide, and hIL-1RalS protein are defined as any native sequence hIL-1RalS or hIL- SRalS variant (which are further defined herein).

A "native sequence hIL-1RalS" means a polypeptide comprising a naturally-occurring S amino acid sequence selected from the group consisting of: the amino acid sequence of 35 amino acid residues from at or about 1 to at or about 38 of Figure 16 (SEQ ID NO:21); the *amino acid sequence of amino acid residues from at or about 26 to at or about 167 of Figure 16 (SEQ ID NO:21); the amino acid sequence of amino acid residues from at or about 39 to at or about 167 of Figure 16 (SEQ ID NO:21); the amino acid sequence of amino acid residues from at or about 47 to at or about 167 of Figure 16 (SEQ ID NO:21); and the amino acid sequence of any naturally-occurring truncated or secreted form or any naturallyoccurring allelic variant of a polypeptide comprising the amino acid sequence of or or or In one embodiment of the invention, the native sequence hIL-1RalS comprises amino acids from at or about 26 to at or about 167 of Figure 16 (SEQ ID NO:21), or amino acids from at or about 1 to at or about 167 of Figure 16 (SEQ ID NO:21). In another embodiment, the native sequence hIL-1RalS consists of amino acids from at or about 47 to at or about 167 of Figure 16 (SEQ ID NO:21) or amino acids from at or about 39 to at or about 167 of Figure 16 (SEQ ID NO:21).

"hIL-lRalS fusion variant" and "hIL-1RalS variant" mean a chimeric hIL-1RalS consisting of a native sequence hIL-1RalS fused at its N-terminus or C-terminus to a heterologous amino acid or amino acid sequence. In one embodiment, the hIL-IRalS fusion variant polypeptide consists of a native sequence of hIL-1RalS fused at its N-terminus or Cterminus to a heterologous amino acid or amino acid sequence, wherein the heterologous amino acid or amino acid sequence is heterologous to the native sequence, i.e. the resulting chimeric sequence is non-naturally occurring. In another embodiment, the hIL-1RalS fusion variant consists of the amino acid sequence of amino acids from at or about 47 to at or about 167 of Figure 16 (SEQ ID NO:21), or the amino acid sequence of amino acids from at or about 39 to at or about 167 of Figure 16 (SEQ ID NO:21), fused at its N-terminus or Cterminus to a heterologous amino acid or amino acid sequence to form a non-naturally occurring fusion protein. Such hIL-1RalS fusion variants include, for instance, hIL-1RalS polypeptides wherein a heterologous secretion leader sequence is fused to the N-terminus of the sequence of amino acid residues from at or about 47 to at or about 167 of Figure 16 (SEQ ID NO:21), or amino acid residues from at or about 39 to at or about 167 of Figure 16 (SEQ ID NO:21).

Human interleukin-1 receptor antagonist analog 2 ("hIL-1Ra2"), hIL-1Ra2 polypeptide, and hIL-1Ra2 protein are defined as any native sequence hIL-1Ra2 or hIL-1Ra2 fusion variant (which are further defined herein).

A "native sequence hIL-lRa2" means a polypeptide comprising the amino acid sequence of amino acid residues from at or about 1 to at or about 134 of Figure 5 (SEQ ID NO: 10) or a polypeptide consisting of a naturally-occurring truncated or secreted form of 3 the polypeptide of In one embodiment of the invention, the native sequence hIL-1Ra2 30 consists of amino acids from at or about 27 to at or about 134 of Figure 5 (SEQ ID NO: 10), or amino acids from at or about 1 to at or about 134 of Figure 5 (SEQ ID NO: "hIL-1Ra2 fusion variant" and "hIL-1Ra2 variant" mean a chimeric hIL-1Ra2 consisting of a native sequence hIL-1Ra2 fused at its N-terminus or C-terminus to a heterologous amino acid or amino acid sequence. In one embodiment, the hIL-1Ra2 fusion 35 variant polypeptide consists of a native sequence of hIL-1Ra2 fused at its N-terminus or Cterminus to a heterologous amino acid or amino acid sequence, wherein the heterologous amino acid or amino acid sequence is heterologous to the native sequence, i.e. the resulting chimeric sequence is non-naturally occurring. In another embodiment, the hIL-1Ra2 variant consists of the amino acid sequence of amino acids from at or about 27 to at or about 134 of Figure 5 (SEQ ID NO:10), or amino acids from at or about 1 to at or about 134 of Figure (SEQ ID NO:10), fused at its N-terminus or C-terminus to a heterologous amino acid or -11amino acid sequence to form a non-naturally occurring fusion protein. Such hIL-1Ra2 fusion variants include, for instance, hIL-1Ra2 polypeptides wherein a heterologous secretion leader sequence is fused to the N-terminus of the sequence of amino acids from at or about 27 to at or about 134 of Figure 5 (SEQ ID NO: 10), or amino acids from at or about 1 to at or about 134 of Figure 5 (SEQ ID NO: Human interleukin-1 receptor antagonist analog 3 ("hIL-lRa3"), hIL-1Ra3 polypeptide, and hIL-1Ra3 protein are defined as any native sequence hIL-1Ra3 or variant hIL-1Ra3 (which are further defined herein).

A "native sequence hIL-1Ra3" means a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 95 to at or about 134 of Figure 7 (SEQ ID NO: 13); the amino acid sequence of amino acid residues from at or about 34 to at or about 155 of Figure 7 (SEQ ID NO: 13); and the amino acid sequence of any naturally-occurring truncated or secreted form or any naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence of or In one embodiment of the invention, the native sequence hIL-1Ra3 comprises amino acids from at or about 34 to at or about 155 of Figure 7 (SEQ ID NO: 13), or amino acids from at or about 2 to at or about 155 of Figure 7 (SEQ ID NO: 13).

"hIL-1Ra3 variant" is defined as any hIL-1Ra3 C-terminal variant or hIL-1Ra3 full sequence variant (which are further defined herein).

"hIL-1Ra3 C-terminal variant" means any hIL-1Ra3 other than a native sequence hIL- 1Ra3, which variant is an active hIL-1Ra3, as defined below, having at least about amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 95 to at or about 134 of Figure 7 (SEQ ID NO: 13) or the amino acid sequence of amino acid residues from at or about 80 to at or about 155 of Figure 7 (SEQ ID NO:13). Such hIL- 1Ra3 C-terminal variants include, for instance, hIL-1Ra3 polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the

S

sequence of amino acid residues from at or about 95 to at or about 134 of Figure 7 (SEQ ID NO: 13) or in the sequence of amino acid residues from at or about 80 to at or about 155 of Figure 7 (SEQ ID NO: 13). Ordinarily, an hIL-1Ra3 C-terminal variant will have at least about amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 95 to at or about 134 of Figure 7 (SEQ ID NO:13) or the amino acid sequence of amino acid residues from at or about 80 to at or about 155 of Figure 7 (SEQ ID NO:13).

35 "hIL-1Ra3 full sequence variant" means any hIL-1Ra3 other than a native sequence hIL-1Ra3, which variant retains at least one biologic activity of a native sequence hIL-1Ra3, such as the ability to bind IL-1R, and which variant has at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about amino acid sequence identity, or at least about 95% amino acid sequence identity with the amino acid sequence of amino acid residues from at or about 34 to at or about 155 of Figure 7 (SEQ ID NO:13) or the amino acid sequence of amino acid residues from at or about 2 to at -12or about 155 of Figure 7 (SEQ ID NO:13). Such hIL-1Ra3 full sequence variants include, for instance, hIL-1Ra3 polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 34 to at or about 155 of Figure 7 (SEQ ID NO:13) or the amino acid sequence of amino acid residues from at or about 2 to at or about 155 of Figure 7 (SEQ ID NO: 13).

Murine interleukin-1 receptor antagonist analog 3 ("mIL-1Ra3"), mIL-1Ra3 polypeptide, and mIL-1Ra3 protein are defined as any native sequence mlL-1Ra3 or variant mIL-1Ra3.

A "native sequence mIL-1Ra3" means a polypeptide comprising an amino acid sequence selected from the group consisting of: the amino acid sequence of amino acid residues from at or about 95 to at or about 134 of Figure 9 (SEQ ID NO: 16); the amino acid sequence of amino acid residues from at or about 34 to at or about 155 of Figure 9 (SEQ ID NO:16); and the amino acid sequence of any naturally-occurring truncated or secreted form or naturally-occurring allelic variant of a polypeptide comprising the amino acid sequence of or In one embodiment of the invention, the native sequence mIL-1Ra3 comprises amino acids from at or about 34 to at or about 155 of Figure 9 (SEQ ID NO: 16).

"mIL-1Ra3 variant" is defined as any mIL-1Ra3 C-terminal variant or mIL-1Ra3 full sequence variant (which are further defined herein).

"mIL-1Ra3 C-terminal variant" means any mIL-1Ra3 other than a native sequence mIL-1Ra3, which variant is an active mIL-1Ra3, as defined below, having at least about amino acid sequence identity with the amino acid sequence of amino acids from at or about to at or about 134 of Figure 9 (SEQ ID NO:16). Such mIL-1Ra3 C-terminal variants include, for instance, mIL-1Ra3 polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acids from at or about 95 to at or about 134 of Figure 9 (SEQ ID NO:16). Ordinarily, an mIL-1Ra3 Cterminal variant will have at least about 80% amino acid sequence identity, or at least about I 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, and or at least about 95% amino acid sequence identity with the amino acid sequence of amino acids 95 to 134 of Figure 9 (SEQ ID NO: 16).

30 "mIL-1Ra3 full sequence variant" means any mIL-1Ra3 other than a native sequence SmIL-1Ra3, which variant retains at least one biologic activity of a native sequence mIL-1Ra3, such as the ability to bind IL-1R, and which variant has at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about sequence identity with the amino acid sequence of amino acid residues from at or about 34 35 to at or about 155 of Figure 9 (SEQ ID NO:16) or the amino acid sequence of amino acid residues from at or about 2 to at or about 155 of Figure 9 (SEQ ID NO:16). Such mIL-1Ra3 full sequence variants include, for instance, mIL-1Ra3 polypeptides wherein one or more amino acid residues are added, or deleted, internally or at the N- or C-terminus, in the sequence of amino acid residues from at or about 34 to at or about 155 of Figure 9 (SEQ ID NO: 16) or in the sequence of amino acid residues from at or about 2 to at or about 155 of Figure 9 (SEQ ID NO:16).

-13- "Human interleukin-1-like polypeptide", "hIL-llp", "hIL-llp polypeptide", "hIL-llp protein", "human interleukin-1 receptor antagonist analog", "hIL-1Raa", "hIL-1Raa polypeptide", and "hIL-iRaa protein" are defined as any hIL-1Ral, hiL-iRa2 or hIL-iRa3 polypeptide.

"Native sequence hIL-llp" and "native sequence hIL-1Raa" are defined as any polypeptide that comprises a native sequence hIL-1Ral, hIL-1Ra2, or hIL-1Ra3.

"hIL-11p variant" is defined as any polypeptide that comprises a variant of hIL-1Ral, hIL-1Ra2, or hIL-1Ra3.

"Interleukin-1 receptor", "interleukin-1 receptor polypeptide", "interleukin-1 receptor protein", "IL-1 receptor", "IL-1R", "IL-1R polypeptide", and "IL-1R protein", are defined as the family of cell surface proteins that bind to interleukin-1 (IL-1) and/or function in IL-1induced signal transduction in a given species, such as human or mouse. IL-1R includes the human T cell-expressed IL-1 receptor disclosed in Sims, et al., Proc. Natl. Acad. Sci. (USA), 86: 8946-8950 (1989).

"Interleukin-18 receptor", "interleukin-18 receptor polypeptide", "interleukin-18 receptor protein", "IL-18 receptor", "IL-18R", "IL-18R polypeptide", and "IL-18R protein", are defined as the family of cell surface proteins that bind to interleukin-18 (IL-18) and/or function in IL-18-induced signal transduction in a given species, such as human or mouse.

IL-18R includes the IL-1 receptor related protein (IL-1Rrp) described in Torigoe et al., J. Biol.

Chem., 272: 25737-25742 (1997) and the IL-18 receptor accessory protein-like molecule (IL- 18RAcPL) described in Born et al., J. Biol. Chem., 273: 29445-29450 (1998).

"Interleukin-1-like family" and "IL-1-like family" are used to indicate the family of polypeptides related to the ligands of IL-1R or IL-18R. The IL-l-like family includes IL-1 receptor agonists and antagonists and related polypeptides such as IL-la (described in Bazan et al., Nature, 379: 591 (1996), IL-11 (Bazan et IL-18 (interferon-( inducing factor)(IGIF)(Bazan et IL-1 receptor antagonist polypeptides such as secretory IL-1Ra (sIL-1Ra)(described in Eisenberg et al., Nature, 343: 341-346 (1990)) and intracellular IL-1Ra (icIL-IRa) (described in Haskill et al. Proc. Natl. Acad. Sci. (USA), 88: 3681-3685 (1991)), and the IL- lp polypeptides of the invention.

30 "Percent amino acid sequence identity" with respect to the IL-llp sequences S* identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in an IL-llp sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence 'W identity, and not considering any conservative substitutions as part of the sequence identity.

35 Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the fulllength of the sequences being compared. For purposes herein, however, amino acid -14sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Tables 3A-3Q. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Tables 3A-3Q has been filed with user documentation in the U.S. Copyright Office, Washington 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Tables 3A-3Q. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which also can be phrased as a given amino acid sequence A that has or comprises a certain amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A to B will not equal the amino acid sequence identity of B to A. As examples of amino acid sequence identity calculations, Tables 2A-2B demonstrate how to calculate the amino acid sequence identity of the amino acid sequence designated "Comparison Protein" to the amino acid sequence designated "PRO".

Unless specifically stated otherwise, all amino acid sequence identity values used Sherein are obtained as described above using the ALIGN-2 sequence comparison computer °program. However, amino acid sequence identity may also be determined using the Ssequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389- 30 3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask yes, strand all, expected occurrences 10, minimum low complexity length 15/5, multi-pass e-value 0.01, constant for multi-pass 25, dropoff for final gapped alignment 25 and 35 scoring matrix BLOSUM62.

In situations where NCBI-BLAST2 is employed for amino acid sequence comparisons, :o:'the amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which also can be phrased as a given amino acid sequence A

S

that has or comprises a certain amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the amino acid sequence identity of A to B will not equal the amino acid sequence identity of B to A.

"Percent nucleic acid sequence identity" with respect to the IL-llp polypeptideencoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in an IL-llp polypeptideencoding nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.

For purposes herein, however, nucleic acid sequence identity values are obtained as described below by using the sequence comparison computer program ALIGN-2, wherein the complete source code for the ALIGN-2 program is provided in Tables 3A-3Q. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code shown in Tables 3A-3Q has been filed with user documentation in the U.S. Copyright Office, Washington 20559, where it is registered under U.S. Copyright Registration No.

TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California or may be compiled from the source code provided in Tables 3A-3Q.

S The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

For purposes herein, the nucleic acid sequence identity of a given nucleic acid i i 30 sequence C to, with, or against a given nucleic acid sequence D (which also can be phrased as a given nucleic acid sequence C that has or comprises a certain nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: *600 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment *0program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity of C to D will not equal the nucleic acid sequence identity of D to C. As examples of nucleic acid sequence identity calculations, Tables 2C-2D demonstrate how to calculate the -16nucleic acid sequence identity of the nucleic acid sequence designated "Comparison DNA" to the nucleic acid sequence designated "PRO-DNA".

Unless specifically stated otherwise, all nucleic acid sequence identity values used herein are obtained as described above using the ALIGN-2 sequence comparison computer program. However, nucleic acid sequence identity may also be determined using the sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res. 25:3389- 3402 (1997)). The NCBI-BLAST2 sequence comparison program may be downloaded from http://www.ncbi.nlm.nih.gov. NCBI-BLAST2 uses several search parameters, wherein all of those search parameters are set to default values including, for example, unmask yes, strand all, expected occurrences 10, minimum low complexity length 15/5, multi-pass e-value 0.01, constant for multi-pass 25, dropoff for final gapped alignment 25 and scoring matrix BLOSUM62.

In situations where NCBI-BLAST2 is employed for sequence comparisons, the nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which also can be phrased as a given nucleic acid sequence C that has or comprises a certain nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program NCBI-BLAST2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the nucleic acid sequence identity of C to D will not equal the nucleic acid sequence identity of D to C.

The term "positives", in the context of the amino acid sequence identity comparisons performed as described above, includes amino acid residues in the sequences compared that are not only identical, but also those that have similar properties. Amino acid residues that 3 score a positive value to an amino acid residue of interest are those that are either identical 30 to the amino acid residue of interest or are a preferred substitution (as defined in Table 1

S.

below) of the amino acid residue of interest.

For purposes herein, the value of positives of a given amino acid sequence A to, with, or against a given amino acid sequence B (which also can be phrased as a given amino acid sequence A that has or comprises a certain positives to, with, or against a given 35 amino acid sequence B) is calculated as follows: a ~100 times the fraction X/Y a a where X is the number of amino acid residues scoring a positive value as defined above by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the -17length of amino acid sequence A is not equal to the length of amino acid sequence B, the positives of A to B will not equal the positives of B to A.

"Isolated," when used to describe the various polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the IL-llp natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

An "isolated" nucleic acid molecule encoding a IL-llp polypeptide is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the IL-llp-encoding nucleic acid. An isolated IL-llp-encoding nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the IL-llp-encoding nucleic acid molecule as it exists in natural cells.

However, an isolated nucleic acid molecule encoding a IL-llp polypeptide includes IL-llpencoding nucleic acid molecules contained in cells that ordinarily express IL-llp where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

25 The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator ;i sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

Nucleic acid is "operably linked" when it is placed into a functional relationship with S another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably 35 linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

-18- The term "antibody" is used in the broadest sense and specifically covers single anti- IL-llp monoclonal antibodies (including agonist, antagonist, and neutralizing antibodies) and anti-IL-11p antibody compositions with polyepitopic specificity. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/O.1% sodium dodecyl sulfate at employ during hybridization a denaturing agent, such as formamide, for example, formamide with 0.1% bovine serum albumin/O. 1% Ficoll/0.1% polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium 25 chloride, 75 mM sodium citrate at 42EC; or employ 50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 0.1% SDS, Sand 10% dextran sulfate at 42EC, with washes at 42EC in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55EC, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at "Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and o*o include the use of washing solution and hybridization conditions temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately 35 stringent conditions is overnight incubation at 37EC in a solution comprising: *I formamide, 5 x SSC (150 mM NaCI, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mL denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50EC. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

-19- The term "epitope tagged" when used herein refers to a chimeric polypeptide comprising an IL-11p polypeptide fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, yet is short enough such that it does not interfere with activity of the polypeptide to which it is fused.

The tag polypeptide preferably also is fairly unique so that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues (preferably, between about 10 and 20 amino acid residues).

As used herein, the term "immunoadhesin" designates antibody-like molecules which combine the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immunoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or igG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM.

"Active" or "activity" for the purposes herein refers to form(s) of IL-llp which retain one or more of the biologic activities of native or naturally-occurring IL-llp, or which exhibit immunological cross-reactivity with a native or naturally-occurring IL-llp.

As used herein, a "biologic activity" or "biological activity" of an IL-llp means any effector function exhibited by the IL-11p in the physiology or pathophysiology of a mammal, excluding any immunogenic or antigenic functions of the IL-llp. Immunogenic and antigenic 25 functions of an IL-llp refer to the ability of the IL-llp to generate a humoral or cell-mediated immune response specific to the IL-llp, and the ability of the IL-llp to specifically recognize o and interact with anti-IL-lip antibodies, B cells or T cells, respectively, in a mammal.

As used herein, "immunological cross-reactivity" with an IL-llp means that the candidate polypeptide is capable of competitively inhibiting the binding of the IL-llp to polyclonal or monoclonal antibodies raised against the IL-llp.

In one embodiment, IL-llp activity includes the ability to agonize or antagonize one or more biological activities of any IL-l-like family member, e.g. an IL-11p activity that antagonizes an IL-1-mediated or IL-18-mediated inflammatory response. In another embodiment, IL-llp activity includes the ability to bind to the IL-18 receptor and/or IL-1 35 receptor.

The term "antagonist" is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native IL-llp polypeptide disclosed herein. In a similar manner, the term "agonist" is used in the broadest sense and includes any molecule that mimics a biological activity of a native IL-11p polypeptide disclosed herein. Suitable agonist or antagonist molecules specifically include agonist or antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native IL-11p polypeptides, peptides, small organic molecules, etc.

"Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

"Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutively done without interruption, but rather is cyclic in nature.

"Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, etc. Preferably, the mammal is human.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

The terms "inflammatory disorders" and "inflammatory diseases" are used interchangeably herein and refer to pathological states resulting in inflammation. Examples of such disorders include inflammatory skin diseases such as psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult 25 respiratory distress syndrome; acute lung injury; Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis; dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; autoimmune diseases such as rheumatoid arthritis, Sjorgen's syndrome, vasculitis, and insulin-dependent diabetes mellitus (IDDM); diseases involving leukocyte diapedesis; central S nervous system (CNS) inflammatory disorder; meningitis; multiple organ injury syndrome secondary to septicaemia or trauma; inflammatory diseases of the liver, including alcoholic hepatitis and hepatic fibrosis; pathologic host responses to infection, including pathologic inflammation in granulomatous diseases, hepatitis, and bacterial pneumonia; antigenantibody complex mediated diseases including glomerulonephritis; sepsis; sarcoidosis; immunopathologic responses to tissue/organ transplantation, including graft-versus host disease (GVHD); inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity .I pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; inflammation in renal diseases, including acute or chronic nephritic conditions such as lupus nephritis; pancreatitis; etc. The preferred indications include rheumatoid arthritis, osteoarthritis, sepsis, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, ischemic reperfusion (including surgical tissue reperfusion injury, stroke, myocardial -21ischemia, and acute myocardial infarction), asthma, psoriasis, graft-versus-host disease (GVHD), and inflammatory bowel disease such as ulcerative colitis.

As used herein, the terms "asthma", "asthmatic disorder", "asthmatic disease", and "bronchial asthma" refer to a condition of the lungs in which there is widespread narrowing of lower airways. "Atopic asthma" and "allergic asthma" refer to asthma that is a manifestation of an IgE-mediated hypersensitivity reaction in the lower airways, including, moderate or severe chronic asthma, such as conditions requiring the frequent or constant use of inhaled or systemic steroids to control the asthma symptoms. A preferred indication is allergic asthma.

II. Detailed Description of the Invention The present invention provides newly identified and isolated nucleotide sequences encoding polypeptides referred to in the present application as IL-llp. In particular, cDNAs encoding IL-llp polypeptides have been identified and isolated, as disclosed in further detail in the Examples below.

Using NCBI-BLAST2 sequence alignment computer programs, it has been found that a full-length native sequence hIL-IRal (shown in Figure 3 and SEQ ID NO:7) has some amino acid sequence identity with human IL-1 receptor antagonist beta (hIL-1Rap) and TANGO-77 protein, a full-length native sequence hIL-1RalL (shown in Figure 15 and SEQ ID NO:19) has some amino acid sequence identity with human IL-1 receptor antagonist beta (hIL-lRap) and TANGO-77 protein, a full-length native sequence hIL-1RalV (shown in Figure 19 and SEQ ID NO:25) has some amino acid sequence identity with human IL-1 receptor antagonist beta (hIL-1Rap) and TANGO-77 protein, a full-length native sequence hIL-1RalS (shown in Figure 16 and SEQ ID NO:21) appears to be an allelic variant of TANGO-77 protein and has some amino acid sequence identity with human IL-1 receptor antagonist beta (hIL- 1Rap), a full-length native sequence hIL-lRa2 (shown in Figure 5 and SEQ ID NO:10) has some amino acid sequence identity with hIL-1Rap, a full-length native sequence hIL-1Ra3 (shown in Figure 7 and SEQ ID NO:13) has some amino acid sequence identity with human intracellular IL-1 receptor antagonist (hicIL-1Ra), and a full-length native sequence mIL-1Ra3 :3 (shown in Figure 9 and SEQ ID NO:16) has some amino acid sequence identity with mouse S 30 IL-1 receptor antagonist (mIL-IRa) and has some amino acid sequence identity with hicIL- IRa. hIL-IRap is described in EP 0855404 published July 29, 1998. TANGO-77 protein is described in WO 99/06426 published February 11, 1999. hicIL-IRa is described in WO S95/10298 published April 20, 1995 and in Haskill et al., Proc. Natl. Acad. Sci. (USA), 88: 3681-3685 (1991). mIL-IRa is described in Zahedi et al., J. Immunol., 146: 4228-4233 35 (1991), Matsushime et al., Blood, 78: 616-623 (1991), Zahedi et al., Cytokine, 6: 1-9 (1994), **Eisenberg et al., Proc. Natl. Acad. Sci. (USA), 88: 5232-5236 (1991) and Shuck et al., Eur. J.

Immunol., 21: 2775-2780 (1991). Accordingly, it is presently believed that the IL-llp polypeptides disclosed in the present application are newly identified members of the interleukin-1-like family and possess inflammatory or anti-inflammatory activities, or other cellular response activating or inhibiting activities, typical of the IL-1-like family.

-22- In addition to the full-length native sequence IL-lip polypeptides described herein, it is contemplated that IL-lip variants can be prepared. Such embodiments of the invention include all IL-ilp polypeptides that are IL-llp variants as defined herein, such as hIL-IRal variants, hIL- 1RalL variants, hIL- 1RalS variants, hIL-l1Ra2 variants, hIL-1Ra3 variants, and mIL-1Ra3 variants.

IL-llp variants can be prepared by introducing appropriate nucleotide changes into the IL-lip DNA, and/or by synthesis of the desired IL-llp polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the ILllp, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

Variations in the native full-length sequence IL-llp or in various domains of the IL-11p described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No.

5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the IL-llp that results in a change in the amino acid sequence of the IL-llp as compared with the native sequence IL-11p. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the IL-llp.

Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the IL-llp with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, conservative amino acid replacements. Insertions or deletions may optionally be in the 25 range of 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the S resulting variants for activity in the in vitro assay described in the Examples below.

Table 1 below lists conservative amino acid substitutions (under the heading of S"Preferred Substitutions") that are useful in generating variants of the native sequence IL- llp.

i30 If such substitutions result in alteration of biological activity, it is useful to introduce more substantial changes, such as the "Exemplary Substitutions" denoted in Table 1 or the o substantial changes described below in reference to amino acid classes, at the active site in @e ~question.

-23- Table 1 Original Exemplary Preferred Residue Substitutions Substitutions Ala val; leu; ile val Arg lys; gin; asn lys Asn gin; his; lys; arg gin Asp glu glu Cys ser ser Gin asn asn Glu asp asp Gly pro; ala ala His asn; gin; lys; arg arg Ile leu; val; met; ala; phe; norleucine leu Leu norleucine; ile; val; met; ala; phe ile Lys arg; gin; asn arg Met leu; phe; ile leu Phe leu; val; ile; ala; tyr leu Pro ala ala Ser thr thr Thr ser ser Trp tyr; phe tyr Tyr trp; phe; thr; ser phe Val ile; leu; met; phe; ala; norleucine leu Substantial modifications in function or immunological identity of the IL-llp polypeptide are accomplished by selecting substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, the charge or hydrophobicity of the *molecule at the target site, or the bulk of the side chain. Naturally occurring residues are 35 divided into groups based on common side-chain properties: hydrophobic: norleucine, met, ala, val, leu, ile; neutral hydrophilic: cys, ser, thr; acidic: asp, glu; basic: asn, gin, his, lys, arg; residues that influence chain orientation: gly, pro; and aromatic: trp, tyr, phe.

**Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

S 45 The variations can be made using methods known in the art such as oligonucleotidemediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zolier et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or -24other known techniques can be performed on the cloned DNA to produce the IL-llp variant

DNA.

Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine.

Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins, Freeman Co., Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

Covalent modifications of IL-llp are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an IL-11p polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the IL-llp. Derivatization with bifunctional agents is useful, for instance, for crosslinking IL-llp to a water-insoluble support matrix or surface for use in the method for purifying anti-IL-11p antibodies, and vice-versa. Commonly used crosslinking agents include, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, Nhydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as methyl-3- [(p-azidophenyl)dithio]propioimidate.

Other modifications include deamidation of glutaminyl and asparaginyl residues to 25 the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the aamino groups of lysine, arginine, and histidine side chains Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman Co., San Francisco, pp. 79-86 (1983)], i* acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

30 Another type of covalent modification of the IL-llp polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.

"Altering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence IL-llp (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic 35 means), and/or adding one or more glycosylation sites that are not present in the native sequence IL-llp. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

Addition of glycosylation sites to the IL-llp polypeptide may be accomplished by altering the amino acid sequence. The alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence IL-llp (for O-linked glycosylation sites). The IL-llp amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the IL-1ip polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on the IL-llp polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the IL-llp polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch.

Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981).

Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

Another type of covalent modification of IL-llp comprises linking the IL-llp polypeptide to one of a variety of nonproteinaceous polymers, polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos.

4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

The IL-llp of the present invention may also be modified in a way to form a chimeric molecule comprising IL-llp fused to another, heterologous polypeptide or amino acid sequence.

S 25 In one embodiment, such a chimeric molecule comprises a fusion of the IL-llp with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.

SThe epitope tag is generally placed at the amino- or carboxyl- terminus of the IL-llp. The presence of such epitope-tagged forms of the IL-llp can be detected using an antibody i against the tag polypeptide. Also, provision of the epitope tag enables the IL-llp to be readily 30 purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.

Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 35 antibodies thereto [Evan et al., Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein S Engineering, 3(6):547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al., Science, 255:192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al., J. Biol. Chem., 266:15163- -26- 15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.

Acad. Sci. USA, 87:6393-6397 (1990)1.

In an alternative embodiment, the chimeric molecule may comprise a fusion of the ILllp with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric molecule (also referred to as an immunoadhesin), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble form of an IL-llp polypeptide in place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.

In one aspect, the invention provides an isolated IL-llp polypeptide that binds to an IL-18R ECD, but not to an IL-1R ECD, the polypeptide having an hIL-1Ral amino acid sequence of residues 37 to 203 of Figure 2 (SEQ ID NO:5), or variants thereof having from 1- 5 additions, deletions or conservative substitutions, or an hIL-1Ral amino acid sequence of residues 15 to 193 of Figure 3 (SEQ ID NO:7) or variants having from 1-5 additions, deletions or conservative substitutions.

The variant preferably has from 1-5 additionally substituted amino acid residues, deleted amino acid residues or 1-5 conservatively substituted residues. More preferably the IL-llp polypeptide cosnsits of the sequence of residues from about 37 to about 203 of Figure 2 (SEQ ID NO:5). Most preferably the IL-11p polypeptide consists of the sequence of residues from about 15 to about 193 of Figure 3 (SEQ ID NO:7).

In another aspect, the invention provides an isolated IL-llp polypeptide that binds to an IL-1R ECD, but not to an IL-18R ECD, in which the polypeptide is an hIL-1RalS 25 polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence of residues 26 to 167 of Figure 16 (SEQ ID NO:21).

9 The sequence identity is preferably 85%, more preferably 90% and most preferably The IL-11p polypeptide may comprise a sequence of residues 26 to 167 of Figure 16 **30 (SEQ ID NO:21).

The IL-11p polypeptide may be provided as a chimeric molecule fused to a heterologous amino acid sequence. The heterologous sequence may be an epitope tag sequence or an Fc region of an immunoglobulin, for example.

Preferred IL-llp polypeptide sequences include residues from 37 to 203 of Figure 2 35 (SEQ ID NO:5), and from 15 to 193 of Figure 3 (SEQ ID NO:7).

In another aspect, the invention provides an isolated polypeptide comprising SEQ ID NO:2.

In another aspect, the invention provides an IL-llp polypeptide consisting of an amino acid sequence selected from the group consisting of: a 26 amino acid IL-llp N-terminal polypeptide encoded by the cDNA insert in the vector deposited as ATCC Dep. No. 203588; -27- H:\Juanita\Keep\patent\25935.4.doc 8/10/04 and a polypeptide encoded by the cDNA insert in the vector deposited as ATCC Dep. No.

203587 the polypeptide binding to an IL-18R ECD but not to an IL-1R ECD, including or alternatively excluding a 36 N-terminal amino acid residue sequence.

In another aspect, the invention provides an IL-llp polypeptide comprising an amino acid sequence that binds to an IL-1R ECD but not to an IL-18R ECD, encoded by the cDNA insert in the vector deposited as ATCC Dep. No. 203855.

In another aspect, the invention provides an isolated DNA molecule encoding an ILllp polypeptide that binds to an IL-18R ECD, but not to an IL-1R ECD, selected from the group consisting of: an hIL-Ral amino acid sequence of residues 37 to 203 of Figure 2 (SEQ ID NO:5) or variants thereof having from 1-5 additions or deletions or conservative substitutions, an hIL-1Ral amino acid sequence of residues 15 to 193 of Figure 3 (SEQ ID NO:7) or variants thereof having from 1-5 additions, deletions or conservative substitutions, and the complement of the DNA molecules of In another aspect, the invention provides an isolated DNA molecule encoding an ILlip polypeptide that binds to an IL-18R ECD, but not to an IL-1R ECD, selected from the group consisting of: an hIL-Ral amino acid sequence of residues 1 to 203 of Figure 2 (SEQ ID NO:5) or variants thereof having from 1-5 additions, deletions or conservative substitutions, an hIL-1Ral amino acid sequence of residues 1 to 193 of Figure 3 (SEQ ID NO:7) or variants thereof having from 1-5 additions, deletions or conservative substitutions, and the complement of the DNA molecules of The DNA molecule encoding an IL-llp polypeptide may encode a variant has from additionally substituted amino acid residues, from 1-5 deleted amino acid residues or from 1-5 conservatively substituted residues.

The DNA molecule may encode an IL-11p polypeptide having a sequence of residues 25 from 37 to 203 of Figure 2 (SEQ ID NO:5), from 15 to 193 of Figure 3 (SEQ ID NO:7), from 1 to 203 of Figure 3 (SEQ ID NO:5) or from 1 to 193 of Figure 3 (SEQ ID NO:7).

In another aspect, the invention provides an isolated DNA molecule encoding an IL- I* lip polypeptide that binds to an IL-1R ECD, but not to an IL-18R ECD, which is an hIL- 1RalS polypeptide comprising an amino acid sequence having at least 80% sequence identity 30 to the sequence of residues 26 to 167 of Figure 16 (SEQ ID NO:21) or the complement of said S DNA molecule.

In another aspect, the invention provides an isolated DNA molecule encoding an ILlip polypeptide that binds to an IL-IR ECD, but not to an IL-18R ECD, which is an hIL- 1RalS polypeptide comprising an amino acid sequence having at least 80% sequence identity 35 to the sequence of residues 1 to 167 of Figure 16 (SEQ ID NO:21) or the complement of said DNA molecule.

The sequence identity is preferably 85%, more preferably 90% and most preferably The isolated DNA molecule may encode an IL-llp polypeptide comprises residues 26 to 167 of Figure 16 (SEQ ID NO:21), or residues 1 to 167 of Figure 16 (SEQ ID NO:21).

-28- H:\Juanita\Keep\patent\25935.4.doc 8/10/04 In another aspect, the invention provides an isolated DNA molecule encoding the ILllp polypeptide sequences including residues from 37 to 203 of Figure 2 (SEQ ID NO:5), and from 15 to 193 of Figure 3 (SEQ ID NO:7) or encoding the polypeptide sequence comprising SEQ ID NO:2.

In another aspect, the invention provides a vector comprising a DNA molecule of the present invention. The vector may further include control sequences recognized by a host cell transfected with the vector, operably linked to a DNA molecule of the present invention.

In another aspect, the invention provides a host cell comprising the vector according to an aspect of the invention.

In another aspect, the invention provides a process for producing an IL-llp polypeptide comprising the steps of culturing a host cell comprising the DNA molecule of the presnet invention under conditions suitable for the expression of the IL-llp polypeptide encoded by the DNA molecule; and recovering said IL-llp polypeptide from the cell culture.

In another aspect, the invention provides an antagonist of IL-lip activity that partially or fully blocks, inhibits or neutralizes binding of a polypeptide according to an aspect of the invention to an IL-18R ECD, which antagonist comprises an antagonist antibody or antibody fragment, or fragments or amino acid sequence variants of native IL-llp polypeptides.

In another aspect, the invention provides an antagonist of IL-llp activity that partially or fully blocks, inhibits or neutralises binding of a polypeptide according to an aspect of the present invention to an IL-1R ECD, which antagonist comprises an antagonist antibody or S.i* antibody fragment, or fragments or amino acid sequence variants of native IL- lp S. polypeptides.

In another aspect, the invention provides an antagonist of IL-llp that partially or fully ".25 blocks, inhibits or neutralises the binding of polypeptides according to the present invention to an IL-1R ECD, which antagonist comprises an antagonist antibody or antibody fragment, fragments or amino acid sequence variants of IL-llp polypeptides.

The antagonist may be an anti-IL-llp antibody, a monoclonal antibody, a human antibody or a humanised antibody.

*30 In another aspect, the invention provides a pharmaceutical composition comprising an IL-llp polypeptide according to an aspect of the present invention, or an antagonist according to an aspect of the present invention, together with a pharmaceutically acceptable carrier.

In another aspect, the invention provides a method of treating an inflammatory disorder disease, comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition according to an aspect of the present invention.

In another aspect, the invention provides for use of an IL-llp polypeptide or an antagonist according to aspects of the present invention in the manufacture of a medicament for use in treating an inflammatory disorder or disease.

-29- H:\Juanita\Keep\patent\25935.4.doc 8/10/04 The inflammatory disorder or disease includes inflammatory skin diseases such as psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis; dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; autoimmune diseases such as rheumatoid arthritis, Sjorgen's syndrome, vasculitis, and insulin-dependent diabetes mellitus (IDDM); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; meningitis; multiple organ injury syndrome secondary to septicaemia or trauma; inflammatory diseases of the liver, including alcoholic hepatitis and hepatic fibrosis; pathologic host responses to infection, including pathologic inflammation in granulomatous diseases, hepatitis, and bacterial pneumonia; antigen-antibody complex mediated diseases including glomerulonephritis; sepsis; sarcoidosis; immunopathologic responses to tissue/organ transplantation, including graft-versus host disease (GVHD); inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; inflammation in renal diseases, including acute or chronic nephritic conditions such as lupus nephritis; pancreatitis.

The inflammatory disorder or disease may be rheumatoid arthritis, osteoarthritis, Ssepsis, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, 25 ischemic reperfusion (including surgical tissue reperfusion injury, stroke, myocardial ischemia, and acute myocardial infarction), asthma, psoriasis, graft-versus-host disease (GVHD), and inflammatory bowel disease such as ulcerative colitis.

The inflammatory disorder or disease may be allergic asthma.

In another aspect, the invention provides a method of treating an IL-1-mediated disorder comprising administering to a mammal in need of such treatment an effective amount of an IL-11p polypeptide according to an aspect of the present invention.

In another aspect, the invention provides for use of an IL-11p polypeptide according to an aspect of the presnt invention in the manufacture of a medicament for use in treating an IL-1-mediated disorder.

In the method or use according to the present invention, the IL-llp polypeptide may be selected from the group consisting of hIL-1Ral, hIL-1RalL, hIL-1RalV, hIL-1RalS, and hIL-1Ra2.

In another aspect, the invention provides a method of treating an IL-18-mediated disorder, comprising administering to a mammal in need of such treatment an effective amount of an IL-llp polypeptide according to an aspect of the present invention.

Ht\Juanita\Keep\patent\25935.4.doc 8/10/04 In another aspect, the invention provides for use of an IL-llp polypeptide according to an aspect of the present invention in the manufacture of a medicament for use in treating an IL-18-mediated disorder.

In the method or use according to the present invention, the IL-11p polypeptide may be selected from the group consisting of hIL-1Ral, hIL-1RalL, hIL-1RalV, hIL-1RalS and hIL-1Ra2. Preferably the IL-11p polypeptide is a native sequence hIL-1Ral.

In another aspect, the invention provides a method of treating an inflammatory disorder, comprising administering to a mammal in need of such treatment an effective amount of an IL-llp according to an aspect of the present invention.

In another aspect, the invention provides for use of an IL-llp according to an aspect of the present invention in the manufacture of a medicament for use in treating an inflammatory disorder.

The IL-11p may be selected from the group consisting of hIL-1Ral and hIL-1RalS, and is preferably a native sequence hIL-1Ral.

The inflammatory condition is preferably asthma, rheumatoid arthritis, osteoarthritis, sepsis, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, ischemic reperfusion disease, such as surgical tissue reperfusion injury, stroke, myocardial ischemia, or acute myocardial infarction, psoriasis, graft-versus-host disease (GVHD) or an inflammatory bowel disease such as ulcerative colitis.

A. Preparation of IL-11p The description below relates primarily to production of IL-11p by culturing cells transformed or transfected with a vector containing IL-llp nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare IL-11p. For instance, the IL-llp sequence, or portions thereof, may be produced by 25 direct peptide synthesis using solid-phase techniques [see, Stewart et al., Solid-Phase SPeptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem.

Soc., 85:2149-2154 (1963)]. In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using manufacturer's 30 instructions. Various portions of the IL-llp may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the full-length IL-llp.

1. Isolation of DNA Encoding IL-llp DNA encoding IL-11p may be obtained from a cDNA library prepared from tissue believed to possess the IL-llp mRNA and to express it at a detectable level. Accordingly, human IL-11p DNA can be conveniently obtained from a cDNA library prepared from human tissue, such as described in the Examples. The IL-l1p-encoding gene may also be obtained from a genomic library or by oligonucleotide synthesis.

Libraries can be screened with probes (such as antibodies to the IL-llp or oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be -31- H:\Juanita\Keep\patent\25935.4.doc 8/10/04 THIS PAGE IS INTENTIONALLY BLANK -32- H:\Juanita\Keep\patent\2593S.4 .doc 8/10/04 conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding iL-iip is to use PCR methodology [Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)].

The Examples below describe techniques for screening a cDNA library. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened.

Methods of labeling are well known in the art, and include the use of radiolabels like 32Plabeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined through sequence alignment using computer software programs such as BLAST, BLAST2, ALIGN-2, DNAstar, and INHERIT which employ various algorithms to measure homology.

Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may i not have been reverse-transcribed into cDNA.

25 2. Selection and Transformation of Host Cells Host cells are transfected or transformed with expression or cloning vectors described :herein for IL-1lp production and cultured in conventional nutrient media modified as S* appropriate for inducing promoters, selecting transformants, or amplifying the genes S* encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In S general, principles, protocols, and practical techniques for maximizing the productivity of cell S cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed.

(IRL Press, 1991) and Sambrook et al., supra.

Methods of transfection are known to the ordinarily skilled artisan, for example, 35 CaP04 and electroporation. Depending on the host cell used, transformation is performed S using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of -33- Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transformations have been described in U.S. Patent No.

4,399,216. Transformations into yeast are typically carried out according to the method of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, polybrene, polyornithine, may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E.

coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635).

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for IL-11p-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism.

Suitable host cells for the expression of glycosylated IL-llp are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells. Examples of useful mammalian host cell lines include Chinese hamster ovary (CHO) and COS cells. More specific examples include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham 25 et al., J. Gen Virol., 36:59 (1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.

.Oo Reprod., 23:243-251 (1980)); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor (MMT 060562, ATCC CCL51). The selection of the appropriate host cell is deemed to be within the skill in the art.

3. Selection and Use of a Replicable Vector **The nucleic acid cDNA or genomic DNA) encoding IL-llp may be inserted into a replicable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by 35 a variety of procedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan.

-34- The IL-llp may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the IL-lip-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No.

5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or supply critical nutrients not available from complex media, the gene encoding D-alanine racemase for Bacilli.

An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the IL-11p-encoding nucleic acid, such as DHFR or thymidine kinase. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is S the trpl gene present in the yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to the ILllp-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a g variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the P-lactamase and lactose promoter systems [Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding IL-11p.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

IL-llp transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus (SV40), from heterologous mammalian promoters, the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

Transcription of a DNA encoding the IL-llp by higher eukaryotes may be increased by 25 inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, o usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, afetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of *the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the IL-1lp coding sequence, but is preferably located at a site from the promoter.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, 35 human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding IL-llp.

-36- Still other methods, vectors, and host cells suitable for adaptation to the synthesis of IL-11p in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620- 625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

4. Detecting Gene Amplification/Expression Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product. Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies may be prepared against a native sequence IL-llp polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to IL-llp DNA and encoding a specific antibody epitope.

Purification of Polypeptide Forms of IL-llp may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable detergent solution Triton-X 100) or by enzymatic cleavage. Cells employed in expression of IL-llp can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents. It may be desired to purify IL-llp from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants S such as IgG; and metal chelating columns to bind epitope-tagged forms of the IL-11p.

Various methods of protein purification may be employed and such methods are known in 35 the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, S Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular IL-llp produced.

B. Activity Assays for IL-11p Variants The biological activity or activities of a particular IL-llp variant polypeptide can be characterized using a variety of in vitro assays known in the art. For example, the ability of -37an hIL-1Ra3 variant polypeptide or a mIL-1Ra3 variant polypeptide to bind IL-1R can be assayed using a radioimmunoprecipitation assay wherein IL-1R extracellular domain (ECD) fused to the Fe region of human immunogiobulin G (IL-1R ECD-Fc) (which can be prepared, as described in Examples 9 and 10 below) is incubated in solution with radiolabeled hIL-1Ra3 variant polypeptide or mIL-1Ra3 variant polypeptide to form labeled complexes, followed by immunoprecipitation of the labeled complexes with goat anti-human IgG Fc and quantitation of radioactivity in the precipitate. In another example, an hIL-1Ra3 variant polypeptide-FLAG tag fusion protein-encoding DNA and an IL-1R ECD-Fc encoding DNA can be coexpressed in a host cell and secreted into the cell's culture medium, followed by immunoprecipitation of culture supernatant with protein G-sepharose and identification of bound hIL-1Ra3 variant polypeptide-FLAG tag fusion protein by immunoblotting with anti- FLAG monoclonal antibody, essentially as described in Example 9 below.

In another embodiment, the ability of an hIL-1Ra3 variant polypeptide or a mIL-1Ra3 variant polypeptide to inhibit the binding of IL-1 to IL-1R can be assayed using a competitive binding assay. For example, a radioimmunoprecipitation assay can be employed wherein IL- 1R ECD-Fc is incubated in solution of radiolabeled IL-1 with or without unlabeled hIL-1Ra3 variant polypeptide or unlabeled mIL-1Ra3 variant polypeptide to form labeled or unlabeled complexes, followed by immunoprecipitation of complexes with anti-human IgG Fc and quantitation of radioactivity in the precipitate. If the presence of unlabeled hIL-1Ra3 variant polypeptide or unlabeled mIL-1Ra3 variant polypeptide in the incubation solution diminishes the radioactivity measured in the resulting immunoprecipitate, the hIL-1Ra3 variant polypeptide or mIL-1Ra3 variant polypeptide in question qualifies as an inhibitor of IL-1 binding to IL-1R. In yet another embodiment, IL-1R ECD-Fc and an hIL-1Ra3 variant-FLAG tag fusion protein or mIL-1Ra3 variant-FLAG tag fusion protein are obtained by recombinant 25 expression in separate cell cultures (essentially as described in Example 10 below), IL-1 and IL-1R ECD-Fc are admixed together with or without the hIL-lRa3 variant-FLAG tag fusion protein or mIL-1Ra3 variant-FLAG tag fusion protein and incubated in solution, the incubation solution is immunoprecipitated with protein G-sepharose, and the bound hIL- 1Ra3 variant-FLAG tag fusion protein or mIL-1Ra3 variant-FLAG tag fusion protein is identified by immunoblotting with anti-FLAG monoclonal antibody. If the presence of IL-1 in the incubation solution diminishes the signal detected by anti-FLAG immunoblotting, the hIL-1Ra3 variant polypeptide or mIL-1Ra3 variant polypeptide in question qualifies as an Sinhibitor of IL-1 binding to IL-1R.

Similarly, the biological activity or activities of a particular hIL-1Ral variant polypeptide can be determined by using a variety of in vitro assays known in the art. For example, the ability of an hIL-Ral variant polypeptide to bind IL-18R can be assayed using a radioimmunoprecipitation assay wherein IL-18R extracellular domain (ECD) fused to the Fc region of human immunoglobulin G (IL-18R ECD-Fc) (which can be prepared, as described in Examples 9 and 10 below) is incubated in solution with radiolabeled hIL-1Ral variant polypeptide to form labeled complex, followed by immunoprecipitation of the labeled complex with goat anti-human IgG Fc and quantitation of radioactivity in the precipitate. In -38another example, an hIL-1Ral variant polypeptide-FLAG tag fusion protein-encoding DNA and an IL-18R ECD-Fc encoding DNA can be coexpressed in a host cell and secreted into the cell's culture medium, followed by immunoprecipitation of culture supernatant with protein G-sepharose and identification of bound hIL-1Ral variant polypeptide-FLAG tag fusion protein by immunoblotting with anti-FLAG monoclonal antibody, essentially as described in Example 9 below.

In another embodiment, the ability of an hIL-1Ral variant polypeptide to inhibit the binding of IL-18 to IL-18R can be assayed using a competitive binding assay. For example, a radioimmunoprecipitation assay can be employed wherein IL-18R ECD-Fc is incubated in solution of radiolabeled IL-18 with or without unlabeled hIL-1Ral variant polypeptide to form labeled or unlabeled complexes, followed by immunoprecipitation of complexes with antihuman IgG Fc and quantitation of radioactivity in the precipitate. If the presence of unlabeled hIL-1Ral variant polypeptide in the incubation solution diminishes the radioactivity measured in the resulting immunoprecipitate, the hIL-1Ral variant polypeptide in question qualifies as an inhibitor of IL-18 binding to IL-18R. In yet another embodiment, IL-18R ECD-Fc and an hIL-1Ral variant-FLAG tag fusion protein are obtained by recombinant expression in separate cell cultures (essentially as described in Example below), IL-18 and IL-18R ECD-Fc are admixed together with or without the hIL-1Ral variant- FLAG tag fusion protein and incubated in solution, the incubation solution is immunoprecipitated with protein G-sepharose, and the bound hIL-1Ral variant-FLAG tag fusion protein is identified by immunoblotting with anti-FLAG monoclonal antibody. If the presence of IL-18 in the incubation solution diminishes the signal detected by anti-FLAG immunoblotting, the hIL-1Ral variant polypeptide in question qualifies as an inhibitor of IL- 18 binding to IL-18R.

S: 25 C. Uses for IL-llp Nucleotide sequences (or their complement) encoding IL-llp have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA. IL-llp nucleic acid will also be useful for the preparation of IL-llp polypeptides by the recombinant techniques described herein.

The full-length native sequence IL-llp genes of Figure 1 (SEQ ID NO:1), Figure 2 (SEQ S ID NO:4), Figure 3 (SEQ ID NO:6), Figure 7 (SEQ ID NO:12), Figure 9 (SEQ ID NO:15) and Figure 16 (SEQ ID NO:20), or portions thereof, may be used as hybridization probes for a cDNA library to isolate the full-length IL-llp gene or to isolate still other genes (for instance, .35 those encoding naturally-occurring variants of IL-l1p or IL-llp from other species) which have a desired sequence identity to the IL-llp sequence disclosed in Figure 1 (SEQ ID NO:1), Figure 2 (SEQ ID NO:4), Figure 3 (SEQ ID NO:6), Figure 7 (SEQ ID NO:12), Figure 9 (SEQ ID NO: 15), or Figure 16 (SEQ ID NO:20). Optionally, the length of the probes will be about 20 to about 50 bases. The hybridization probes may be derived from the nucleotide sequence of Figure 1 (SEQ ID NO:1), Figure 2 (SEQ ID NO:4), Figure 3 (SEQ ID NO:6), Figure 7 (SEQ ID NO:12), Figure 9 (SEQ ID NO:15), or Figure 16 (SEQ ID NO:20), or from genomic sequences -39including promoters, enhancer elements and introns of native sequence IL-llp. By way of example, a screening method will comprise isolating the coding region of the IL-llp gene using the known DNA sequence to synthesize a selected probe of about 40 bases.

Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 32p or 35S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the IL-11p gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA to determine which members of such libraries the probe hybridizes to. Hybridization techniques are described in further detail in the Examples below.

The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related IL-llp coding sequences.

Nucleotide sequences encoding an IL-llp can also be used to construct hybridization probes for mapping the gene which encodes that IL-lip and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions of a chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.

When the coding sequences for IL-llp encode a protein which binds to another protein (example, where the IL-lip binds to an IL-1 receptor or IL-18 receptor), the IL-l11p can be used in assays to identify the other proteins or molecules involved in the binding interaction. By such methods, inhibitors of the receptor/ligand binding interaction can be identified. Proteins involved in such binding interactions can also be used to screen for peptide or small molecule inhibitors or agonists of the binding interaction. Screening assays can be designed to find lead compounds that mimic the biological activity of a native IL-llp or a receptor for IL-11p. Such screening assays will include assays amenable to highthroughput screening of chemical libraries, making them particularly suitable for identifying small molecule drug candidates. Small molecules contemplated include synthetic organic or S inorganic compounds. The assays can be performed in a variety of formats, including •i S protein-protein binding assays, biochemical screening assays, immunoassays and cell based 30 assays, which are well characterized in the art.

Nucleic acids which encode IL-11p or its modified forms can also be used to generate either transgenic animals or "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal a 0o* mouse or rat) is an animal having cells that contain a transgene, which transgene was 35 introduced into the animal or an ancestor of the animal at a prenatal, an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding IL-llp can be used to clone genomic DNA encoding IL-llp in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express DNA encoding IL-l11p. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S.

Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for IL-llp transgene incorporation with tissue-specific enhancers. Transgenic animals that include a copy of a transgene encoding IL-lip introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of DNA encoding IL-11p. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression. In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.

Alternatively, non-human homologues of IL-11p can be used to construct an IL-llp "knock out" animal which has a defective or altered gene encoding IL-llp as a result of homologous recombination between the endogenous gene encoding IL-11p and altered genomic DNA encoding IL-11p introduced into an embryonic cell of the animal. For example, cDNA encoding IL-11p can be used to clone genomic DNA encoding IL-11p in accordance with established techniques. A portion of the genomic DNA encoding IL-llp can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the and 3' ends) are included in the vector [see Thomas and Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see Li et al., Cell, 69:915 (1992)]. The selected cells are then injected into a blastocyst of an animal a mouse or rat) to form aggregation chimeras [see Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113- 25 152]. A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for *:30 their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the IL-llp polypeptide.

Nucleic acid encoding the IL-llp polypeptides may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, S which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane.

-41- (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83, 4143-4146 [1986]). The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.

There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnology 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibody specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g. capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptormediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et al., Science 256, 808-813 (1992).

The IL-llp polypeptides of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the IL-llp product hereof is combined in admixture with a pharmaceutically acceptable carrier vehicle.

.25 Therapeutic formulations are prepared for storage by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and 30 include buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, S* such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as S polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or '35 dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; saltg forming counterions such as sodium; and/or nonionic surfactants such as Tween, Pluronics or PEG.

The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or following lyophilization and reconstitution.

-42- Therapeutic compositions herein generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is in accord with known methods, e.g. injection or infusion by intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial or intralesional routes, topical administration, or by sustained release systems.

Dosages and desired drug concentrations of pharmaceutical compositions of the present invention may vary depending on the particular use envisioned. The determination of the appropriate dosage or route of administration is well within the skill of an ordinary physician. Animal experiments provide reliable guidance for the determination of effective doses for human therapy. Interspecies scaling of effective doses can be performed following the principles laid down by Mordenti, J. and Chappell, W. "The use of interspecies scaling in toxicokinetics" In Toxicokinetics and New Drug Development, Yacobi et al., Eds., Pergamon Press, New York 1989, pp. 42-96.

An "effective amount" of the IL-llp to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the patient. Accordingly, it will be necessary for the therapist to titer the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. Typically, the clinician will administer the IL-llp until a dosage is reached that achieves the desired effect. The progress of this therapy is easily monitored by conventional assays.

D. Anti-IL-llp Antibodies The present invention further provides anti-IL-llp antibodies. Exemplary antibodies include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies.

1. Polyclonal Antibodies 25 The anti-IL-llp antibodies may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal *i by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the IL-llp polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.

S Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.

2. Monoclonal Antibodies The anti-IL-llp antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other -43appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent will typically include the IL-llp polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].

Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.

The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland.

Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); 25 Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63].

The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against IL-llp. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by 30 immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbant assay (ELISA). Such techniques and assays are known in the S art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods [Goding, supra]. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures -44such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The monocional antibodies may also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also may be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [U.S.

Patent No. 4,816,567; Morrison et al., supra] or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigencombining site of an antibody of the invention to create a chimeric bivalent antibody.

The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.

Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.

25 In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.

3. Human and Humanized Antibodies The anti-IL-llp antibodies of the invention may further comprise humanized *30 antibodies or human antibodies. Humanized forms of non-human murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, S* Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain S minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary *,35 determining region (CDR) of the recipient are replaced by residues from a CDR of a nonhuman species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region typically that of a human immunoglobulin [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2: 593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain.

Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991)]. The techniques of Cole et al. and Boerner et al.

are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 25 147(1): 86-95 (1991)]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., 0 Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar, Intern. Rev. Immunol.

35 13 65-93 (1995).

4. Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for the IL-llp, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit.

-46- Methods for making bispecific antibodies are known in the art. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunogiobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published 13 May 1993, and in Traunecker et al., EMBO 10: 3655-3659 (1991).

Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are cotransfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

Heteroconiugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention.

Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells Patent No. 4,676,980], and for treatment of HIV infection [WO 91/00360; WO 92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using 25 known methods in synthetic protein chemistry, including those involving crosslinking agents.

For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S.

Patent No. 4,676,980.

30 E. Uses for anti-IL-llp Antibodies The anti-IL-llp antibodies of the invention have various utilities. For example, anti- IL-llp antibodies may be used in diagnostic assays for IL-llp, detecting its expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158].

The antibodies used in the diagnostic assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as 3 H, 14C, 32p, 3S, or 1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or -47horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144: 945 (1962); David et al., Biochemistri, 13: 1014 (1974); Pain et al., J. Immunoi.

Meth., 40: 219 (1981); and Nygren, J. Histochem. and Cytochem., 30: 407 (1982).

Anti-IL-llp antibodies also are useful for the affinity purification of IL-llp from recombinant cell culture or natural sources. In this process, the antibodies against IL-llp are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containing the IL-11p to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the IL-llp, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the IL-llp from the antibody.

In addition, anti-IL-llp antibodies are useful as therapeutic agents for targeting of native IL-llp in IL-11p-mediated disease conditions, e.g. disease states characterized by pathologic IL-1 or IL-18 agonist or agonist-like activity of the native IL-11p. In the treatment and prevention of a native IL-11p-mediated disorder with the anti-IL-llp antibody of the invention, the antibody composition will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the antibody, the particular type of antibody, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The "effective amount" or "therapeutically effective amount" of antibody to be administered will be governed by such considerations, and is the minimum amount necessary to prevent, ameliorate, or treat the native IL-1lp- 25 mediated disorder, including treating inflammatory diseases and reducing inflammatory responses. Such amount is preferably below the amount that is toxic to the host or renders the host significantly more susceptible to infections.

As a general proposition, the initial pharmaceutically effective amount of the antibody or antibody fragment administered parenterally per dose will be in the range of about 0.1 to *30 50 mg/kg of patient body weight per day, with the typical initial range of antibody used being 0.3 to 20 mg/kg/day, more preferably 0.3 to 15 mg/kg/day.

In one embodiment, using systemic administration, the initial pharmaceutically effective amount will be in the range of about 2 to 5 mg/kg/day.

For methods of the invention using administration by inhalation, the initial 35 pharmaceutically effective amount will be in the range of about 1 microgram (:g)/kg/day to 100 mg/kg/day.

In one embodiment, the invention provides a method for treating an IL-11p-mediated inflammatory disorder comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

-48- In another embodiment, the invention provides a method for treating an hIL-llpmediated asthmatic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-lip antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated asthmatic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ral antibody.

In another embodiment, the invention provides a method for treating an IL-lipmediated rheumatoid arthritic disorder comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-lipmediated rheumatoid arthritic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated rheumatoid arthritic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-lRal antibody.

In another embodiment, the invention provides a method for treating an IL-lipmediated osteoarthritic disorder comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-llpmediated osteoarthritic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated osteoarthritic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ral antibody.

In another embodiment, the invention provides a method for treating an IL-llpmediated septic disorder comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-llpmediated septic disorder comprising administering to a human in need of such treatment an 30 effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated septic disorder comprising administering to a human in need of such treatment an S effective amount of an anti-hIL-1Ral antibody.

In another embodiment, the invention provides a method for treating IL-llp-mediated 35 acute lung injury comprising administering to a mammal, such as human, in need of such S treatment an effective amount of an anti-IL- lp antibody.

In another embodiment, the invention provides a method for treating hIL-llpmediated acute lung injury comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

-49- In another embodiment, the invention provides a method for treating hIL-IRalmediated acute lung injury comprising administering to a human in need of such treatment an effective amount of an anti-hiL-iRal antibody.

In another embodiment, the invention provides a method for treating IL-lip-mediated adult respiratory distress syndrome comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating hIL-11pmediated adult respiratory distress syndrome comprising administering to a human in need of such treatment an effective amount of an anti-hIL- llp antibody.

In another embodiment, the invention provides a method for treating hIL-lRalmediated adult respiratory distress syndrome comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ra 1 antibody.

In another embodiment, the invention provides a method for treating IL-11p-mediated idiopathic pulmonary fibrosis comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating hIL-llpmediated idiopathic pulmonary fibrosis comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating hIL-1Ralmediated idiopathic pulmonary fibrosis comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ral antibody.

In another embodiment, the invention provides a method for treating an IL-lIpmediated ischemic reperfusion disease, such as surgical tissue reperfusion injury, stroke, myocardial ischemia, or acute myocardial infarction, comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL- llp antibody.

In another embodiment, the invention provides a method for treating an hIL-llpmediated ischemic reperfusion disease, such as surgical tissue reperfusion injury, stroke, myocardial ischemia, or acute myocardial infarction, comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

30 In another embodiment, the invention provides a method for treating an hIL-iRalmediated ischemic reperfusion disease, such as surgical tissue reperfusion injury, stroke, myocardial ischemia, or acute myocardial infarction, comprising administering to a human in need of such treatment an effective amount of an anti-hIL-lRal antibody.

SIn another embodiment, the invention provides a method for treating an IL-llp- 35 mediated psoriatic disorder comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-11p antibody.

In another embodiment, the invention provides a method for treating an hIL-11pmediated psoriatic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-lRalmediated psoriatic disorder comprising administering to a human in need of such treatment an effective amount of an anti-hIL- Ral antibody.

In another embodiment, the invention provides a method for treating an IL-11pmediated graft-versus-host disease (GVHD) comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-11pmediated graft-versus-host disease (GVHD) comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated graft-versus-host disease (GVHD) comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ra antibody.

In another embodiment, the invention provides a method for treating an IL-llpmediated inflammatory bowel disease such as ulcerative colitis, comprising administering to a mammal, such as human, in need of such treatment an effective amount of an anti-IL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-11pmediated inflammatory bowel disease such as ulcerative colitis, comprising administering to a human in need of such treatment an effective amount of an anti-hIL-llp antibody.

In another embodiment, the invention provides a method for treating an hIL-1Ralmediated inflammatory bowel disease such as ulcerative colitis, comprising administering to a human in need of such treatment an effective amount of an anti-hIL-1Ral antibody.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified 30 in the following examples, and throughout the specification, by ATCC accession numbers is the American Type Culture Collection, Manassas, VA.

EXAMPLE 1 Isolation of DNA encoding hIL-1Ral and mIL-1Ra3 A public expressed sequence tag (EST) DNA database (Genbank) was searched with i 35 human interleukin-1 receptor antagonist (hIL-IRa) sequence, also known as secretory human interleukin-1 receptor antagonist ("sIL-IRa") sequence, and a human EST designated AI014548 (Figure 4, SEQ ID NO:8), and a murine EST designated W08205 (Figure 10, SEQ ID NO:17), were identified, which showed homology with the known protein hIL-1Ra (sIL- 1 Ra).

EST clones AI014548 and W08205 were purchased from Research Genetics (Huntsville,AL) and the cDNA inserts were obtained and sequenced in their entireties.

-51- The entire nucleotide sequence of the clone AI014548, designated DNA85066, is shown in Figure 1 (SEQ ID NO:1). Clone DNA85066 contains a single open reading frame that is interrupted by an apparent intronic sequence. The intron is bounded by splice junctions at nucleotide positions 181 to 186 (splice donor site) and nucleotide positions 430 to 432 (splice acceptor site) (Fig. 1; SEQ ID NO: 1).

A virtual processed nucleotide sequence (Fig. 3; SEQ ID NO:6), designated DNA94618, was derived by removing the apparent intronic sequence from clone DNA85066. Clone DNA94618 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 103-105, and a stop codon at nucleotide positions 682-684 (Fig. 3; SEQ ID NO:6). The predicted polypeptide precursor (hIL-lRal) (Fig. 3; SEQ ID NO:7) is 193 amino acids long. The putative signal sequence extends from amino acid positions 1 to 14. A putative cAMP- and cGMP-dependent protein kinase phosphorylation site is located at amino acid positions 33-36. Putative N-myristoylation sites are located at amino acid positions and 87-92.

Clone DNA85066 (designated as DNA85066-2534) has been deposited with ATCC and was assigned ATCC deposit no. 203588. The full-length hIL-lRal protein shown in Figure 3 (SEQ ID NO:7) has an estimated molecular weight of about 21,822 daltons and a pl of about 8.9.

Based on a sequence alignment analysis of the full-length sequence (SEQ ID NO:7), hIL-iRal shows significant amino acid sequence identity to hIL-IRa (sIL-iRa) and hIL-lRap proteins.

The entire nucleotide sequence of the clone W08205, designated DNA92505, is shown in Figure 9 (SEQ ID NO:15). Clone DNA92505 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 145-147, and a stop codon at nucleotide positions 610-612 (Fig. 9; SEQ ID NO:15). The predicted polypeptide S. precursor (mIL-lRa3) (Fig. 9; SEQ ID NO:16) is 155 amino acids long. The putative signal S sequence extends from amino acid positions 1-33. Putative N-myristoylation sites are located at amino acid positions 29-34, 60-65, 63-68, 91-96 and 106-111. An interleukin-l-like sequence is located at amino acid positions 111-131.

30 Clone DNA92505 (designated as DNA92505-2534) was deposited with ATCC and was assigned ATCC deposit no. 203590. The full length mIL-1Ra3 protein shown in Figure 9 (SEQ ID NO: 16) has an estimated molecular weight of about 17,134 daltons and a pi of about 4.8.

Based on a sequence alignment analysis of the full-length sequence (SEQ ID NO: 16), 35 mIL-1Ra3 shows significant amino acid sequence identity to mIL-1Ra, hicIL-1Ra, hIL-IRa (sIL-1Ra) and hIL-1Rap proteins.

EXAMPLE 2 Isolation of DNA encoding hIL-lra2 and hI, -1Ra3 A expressed sequence tag (EST) DNA database (LIFESEQ®, Incyte Pharmaceuticals, Palo Alto, CA) was searched with human interleukin-1 receptor antagonist (hIL-1Ra) -52sequence, also known as secretory human interleukin-1 receptor antagonist ("sIL-1Ra") sequence, and the ESTs, designated 1433156 (Figure 5, SEQ ID NO:9) and 5120028 (Figure 7, SEQ ID NO:12), were identified, which showed homology with the hIL-1Ra known protein.

EST clones 1433156 and 5120028 were purchased from Incyte Pharmaceuticals (Palo Alto, CA) and the cDNA inserts were obtained and sequenced in their entireties.

The entire nucleotide sequence of the clone 1433156, designated DNA92929, is shown in Figure 5 (SEQ ID NO:9). Clone DNA92929 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 96-98, and a stop codon at nucleotide positions 498-500 (Fig. 5; SEQ ID NO:9). The predicted polypeptide precursor (hIL-1Ra2) (Fig. 5; SEQ ID NO:10) is 134 amino acids long. A putative signal sequence extends from amino acid positions 1-26.

Clone DNA92929 (designated as DNA92929-2534) was deposited with ATCC and was assigned ATCC deposit no. 203586. The full-length hIL-lRa2 protein shown in Figure (SEQ ID NO: 10) has an estimated molecular weight of about 14,927 daltons and a pi of about 4.8.

Based on a sequence alignment analysis of the full-length sequence (SEQ ID hIL-1Ra2 shows significant amino acid sequence identity to hIL-1Rap protein. hIL-1Ra2 is believed to be a splice variant of hIL-1Rap.

The entire nucleotide sequence of the clone 5120028, designated DNA96787, is shown in Figure 7 (SEQ ID NO:12). Clone DNA96787 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 1-3, and a stop codon at nucleotide positions 466-468 (Fig. 7; SEQ ID NO:12). The predicted polypeptide precursor (hIL-1Ra3) (Fig. 7; SEQ ID NO:13) is 155 amino acids long. A putative signal sequence extends from amino acid positions 1-33. Putative N-myristoylation sites are located at amino acid positions 29-34, 60-65, 63-68, 73-78, 91-96 and 106-111. An interleukin-1-like sequence is located at amino acid positions 111-131.

It is believed that the predicted 155 amino acid polypeptide of hIL-1Ra3 behaves as a mature sequence (without a presequence that is removed in post-translational processing) in certain animal cells. It is also believed that other animal cells recognize and remove one or more signal peptide(s) extending from amino acid positions 1 to about 33. As shown in Example 14 below, transiently transfected CHO host cells secrete a form of hIL-lRa3 that only lacks the N-terminal methionine in the sequence of Figure 7 (SEQ ID NO:13).

Clone DNA96787 (designated as DNA96787-2534) was deposited with ATCC and was assigned ATCC deposit no. 203589. The full length hIL-1Ra3 protein shown in Figure 7 (SEQ 35 ID NO:13) has an estimated molecular weight of about 16,961 daltons and a pi of about 4.9.

Based on a sequence alignment analysis of the full-length sequence (SEQ ID NO: 13), hIL-1Ra3 shows significant amino acid sequence identity to hicIL-1Ra and hIL-1Ra (sIL-1Ra) proteins.

EXAMPLE 3 Northern Blot Analysis -53- Expression of hIL-1Ra3 mRNA in human tissues and mIL-lRa3 mRNA in mouse tissues was examined by Northern blot analysis. Human and mouse multiple tissue northern (RNA) blots and mouse embryo blots were purchased from Ciontech and probed with corresponding cDNA according to the manufacturer's instructions.

As shown in Fig. 11, hIL-1Ra3 mRNA (2.7 kb) were detected only in human placenta and mIL-1Ra3 mRNA transcripts (1.4 kb and 2.5 kb) were detected only in the day-17 mouse embryo.

EXAMPLE 4 Use of IL-llp as a hybridization probe The following method describes use of a nucleotide sequence encoding IL-llp as a hybridization probe.

DNA comprising the coding sequence of full-length IL-llp (as shown in Figures 3, 7, 9, 15, 16 and 19; SEQ ID NOS:6, 9, 12, 15, 18, 20 and 24) is employed as a probe to screen for homologous DNAs (such as those encoding naturally-occurring variants of IL-llp) in human tissue cDNA libraries or human tissue genomic libraries.

Hybridization and washing of filters containing either library DNAs is performed under the following high stringency conditions. Hybridization of radiolabeled IL-11p-derived probe to the filters is performed in a solution of 50% formamide, 5x SSC, 0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH 6.8, 2x Denhardt's solution, and dextran sulfate at 420C for 20 hours. Washing of the filters is performed in an aqueous solution of 0. lx SSC and 0.1% SDS at 42oC.

DNAs having a desired sequence identity with the DNA encoding full-length native sequence IL-llp can then be identified using standard techniques known in the art.

EXAMPLE Expression of IL-11p in E. coli This example illustrates preparation of an unglycosylated form of IL-llp by S recombinant expression in E. coli.

The DNA sequence encoding an IL-llp is initially amplified using selected PCR primers. The primers should contain restriction enzyme sites which correspond to the 30 restriction enzyme sites on the selected expression vector. A variety of expression vectors Smay be employed. An example of a suitable vector is pBR322 (derived from E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillin and tetracycline resistance. The vector is digested with restriction enzyme and dephosphorylated. The PCR amplified sequences are then ligated into the vector. The vector will preferably include 35 sequences which encode for an antibiotic resistance gene, a trp promoter, a polyhis leader o* (including the first six STII codons, polyhis sequence, and enterokinase cleavage site), the ILlip coding region, lambda transcriptional terminator, and an argU gene.

The ligation mixture is then used to transform a selected E. coli strain using the methods described in Sambrook et al., supra. Transformants are identified by their ability to grow on LB plates and antibiotic resistant colonies are then selected. Plasmid DNA can be isolated and confirmed by restriction analysis and DNA sequencing.

-54- Selected clones can be grown overnight in liquid culture medium such as LB broth supplemented with antibiotics. The overnight culture may subsequently be used to inoculate a larger scale culture. The cells are then grown to a desired optical density, during which the expression promoter is turned on.

After culturing the cells for several more hours, the cells can be harvested by centrifugation. The cell pellet obtained by the centrifugation can be solubilized using various agents known in the art, and the solubilized IL-llp protein can then be purified using a metal chelating column under conditions that allow tight binding of the protein.

EXAMPLE 6 Expression of IL-llp in mammalian cells This example illustrates preparation of a potentially glycosylated form of IL-11p by recombinant expression in mammalian cells.

The vector, pRK5 (see EP 307,247, published March 15, 1989), is employed as the expression vector. Optionally, the IL-11p DNA is ligated into pRK5 with selected restriction enzymes to allow insertion of the IL-llp DNA using ligation methods such as described in Sambrook et al., supra. The resulting vector is called In one embodiment, the selected host cells may be 293 cells. Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics.

About 10 pg pRK5-IL-llp DNA is mixed with about 1 pg DNA encoding the VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)) and dissolved in 500 p1 of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl2. To this mixture is added, dropwise, 500 l1 of 50 mM HEPES (pH 7.35), 280 mM NaCI, 1.5 mM NaPO 4 and a precipitate is allowed to form for 10 minutes at The precipitate is suspended and added to the 293 cells and allowed to settle for about four hours at 37oC. The culture medium is aspirated off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293 cells are then washed with serum free medium, fresh S• medium is added and the cells are incubated for about 5 days.

Approximately 24 hours after the transfections, the culture medium is removed and replaced with culture medium (alone) or culture medium containing 200 pCi/ml 3 5 S-cysteine 30 and 200 pCi/ml 3 5 S-methionine. After a 12 hour incubation, the conditioned medium is collected, concentrated on a spin filter, and loaded onto a 15% SDS gel. The processed gel S may be dried and exposed to film for a selected period of time to reveal the presence of IL-llp polypeptide. The cultures containing transfected cells may undergo further incubation (in serum free medium) and the medium is tested in selected bioassays.

35 In an alternative technique, IL-11p may be introduced into 293 cells transiently using the dextran sulfate method described by Somparyrac et al., Proc. Natl. Acad. Sci. USA, 12: S7575 (1981). 293 cells are grown to maximal density in a spinner flask and 700 pg Slip DNA is added. The cclls are first concentrated from the spinner flask by centrifugation and washed with PBS. The DNA-dextran precipitate is incubated on the cell pellet for four hours. The cells are treated with 20% glycerol for 90 seconds, washed with tissue culture medium, and re-introduced into the spinner flask containing tissue culture medium, 5 :g/ml bovine insulin and 0.1 pg/ml bovine transferrin. After about four days, the conditioned media is centrifuged and filtered to remove cells and debris. The sample containing expressed IL-llp can then be concentrated and purified by any selected method, such as dialysis and/or column chromatography.

In another embodiment, IL-llp can be expressed in CHO cells. The pRK5-IL-llp can be transfected into CHO cells using known reagents such as CaPO 4 or DEAE-dextran. As described above, the cell cultures can be incubated, and the medium replaced with culture medium (alone) or medium containing a radiolabel such as 3 5 S-methionine. After determining the presence of IL-llp polypeptide, the culture medium may be replaced with serum free medium. Preferably, the cultures are incubated for about 6 days, and then the conditioned medium is harvested. The medium containing the expressed IL-11p can then be concentrated and purified by any selected method.

Epitope-tagged IL-llp may also be expressed in host CHO cells. The IL-llp may be subcloned out of the pRK5 vector. The subclone insert can undergo PCR to fuse in frame with a selected epitope tag such as a poly-his tag into a Baculovirus expression vector. The poly-his tagged IL-llp insert can then be subcloned into a SV40 driven vector containing a selection marker such as DHFR for selection of stable clones. Finally, the CHO cells can be transfected (as described above) with the SV40 driven vector. Labeling may be performed, as described above, to verify expression. The culture medium containing the expressed poly-His tagged IL-llp can then be concentrated and purified by any selected method, such as by Ni 2 chelate affinity chromatography.

EXAMPLE 7 Expression of IL-llp in Yeast 25 The following method describes recombinant expression of IL-llp in yeast.

First, yeast expression vectors are constructed for intracellular production or 'secretion of IL-llp from the ADH2/GAPDH promoter. DNA encoding IL-llp and the promoter is inserted into suitable restriction enzyme sites in the selected plasmid to direct intracellular expression of IL-llp. For secretion, DNA encoding IL-11p can be cloned into the selected 30 plasmid, together with DNA encoding the ADH2/GAPDH promoter, a native IL-llp signal peptide or other mammalian signal peptide, or, for example, a yeast alpha-factor or invertase secretory signal/leader sequence, and linker sequences (if needed) for expression of IL-llp.

Yeast cells, such as yeast strain AB110, can then be transformed with the expression plasmids described above and cultured in selected fermentation media. The transformed *35 yeast supernatants can be analyzed by precipitation with 10% trichloroacetic acid and .separation by SDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

Recombinant IL-llp can subsequently be isolated and purified by removing the yeast cells from the fermentation medium by centrifugation and then concentrating the medium using selected cartridge filters. The concentrate containing IL-11p may further be purified using selected column chromatography resins.

-56- EXAMPLE 8 Expression of IL-llp in Baculovirus-Infected Insect Cells The following method describes recombinant expression of IL-11p in Baculovirusinfected insect cells.

The sequence coding for IL-llp is fused upstream of an epitope tag contained within a baculovirus expression vector. Such epitope tags include poly-his tags and immunoglobulin tags (like Fc regions of IgG). A variety of plasmids may be employed, including plasmids derived from commercially available plasmids such as pVL1393 (Novagen). Briefly, the sequence encoding IL-llp or the desired portion of the coding sequence of IL-11p (such as the sequence encoding the mature protein) is amplified by PCR with primers complementary to the 5' and 3' regions. The 5' primer may incorporate flanking (selected) restriction enzyme sites. The product is then digested with those selected restriction enzymes and subcloned into the expression vector.

Recombinant baculovirus is generated by co-transfecting the above plasmid and BaculoGoldTM virus DNA (Pharmingen) into Spodoptera frugiperda cells (ATCC CRL 1711) using lipofectin (commercially available from GIBCO-BRL). After 4 5 days of incubation at 28oC, the released viruses are harvested and used for further amplifications.

Viral infection and protein expression are performed as described by O'Reilley et al., Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford University Press (1994).

Expressed poly-his tagged IL-11p can then be purified, for example, by Ni 2 -chelate affinity chromatography as follows. Extracts are prepared from recombinant virus-infected Sf9 cells as described by Rupert et al., Nature, 362: 175-179 (1993). Briefly, Sf9 cells are washed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mM MgCl 2 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KC1), and sonicated twice for 20 seconds on ice. The sonicates are cleared by centrifugation, and the supernatant is diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCI, 10% glycerol, pH 7.8) and filtered through a 0.45 (Dm filter. A Ni2*-NTA agarose column (commercially available from Qiagen) is prepared with a bed volume of 5 mL, washed with 25 mL of water and equilibrated with 25 mL of loading buffer. The filtered cell extract is loaded onto the column at 0.5 mL per minute. The column 30 is washed to baseline A 2 8 0 with loading buffer, at which point fraction collection is started.

SNext, the column is washed with a secondary wash buffer (50 mM phosphate; 300 mM NaCl, glycerol, pH which elutes nonspecifically bound protein. After reaching A 280 baseline again, the column is developed with a 0 to 500 mM Imidazole gradient in the secondary wash buffer. One mL fractions are collected and analyzed by SDS-PAGE and 5* 35 silver staining or Western blot with Ni2+-NTA-conjugated to alkaline phosphatase (Qiagen).

S Fractions containing the eluted Hisio-tagged IL-llp are pooled and dialyzed against loading buffer.

Alternatively, purification of the IgG tagged (or Fc tagged) IL-llp can be performed using known chromatography techniques, including for instance, Protein A or protein G column chromatography.

EXAMPLE 9 -57- IL-18 Receptor and IL-1 Receptor Binding of hIL-1Ral To facilitate the characterization of hIL-1Ral, a PCR fragment containing the partial ORF of clone DNA85066 (Figure 1; SEQ ID NO:3) was cloned into pCMV1FLAG (IBI Kodak, described in Pan et al., Science, 276: 111-113) as an in-frame fusion to a NH2-terminal preprotrypsin leader sequence and FLAG tag encoded by the vector. The entire cDNA insert of the recombinant pCMV1FLAG vector clone (designated clone DNA96786) was sequenced (Figure 2; SEQ ID NO:4). The cDNAs encoding the extracellular domain of hILlR and hIL18R (formerly known as hILlRrp) were obtained by polymerase chain reaction (PCR) and cloned into a modified pCMV1FLAG vector that allowed for in-frame fusion with the Fc portion of human immunoglobulin G.

Human embryonic kidney 293 cells were grown in high glucose DMEM (Genentech, Inc). The cells were seeded at 3-4 X10 6 per plate (100 mm) and co-transfected with pCMV1FLAG-hIL-1Ral and pCMV1FLAG-ILIR-ECD-Fc or pCMV1FLAG-IL18R-ECD-Fc by means of calcium phosphate precipitation. The media were changed 12 hours post transfection. The resultant conditioned media (10 ml each) were harvested after a further 74 hour incubation, clarified by centrifugation, aliquoted and stored at -70°C. The receptor- Fc and ligand complex from 1.5 ml conditioned medium was immunoprecipitated with protein G-Sepharose, washed three times with buffer containing 50 mM Hepes, pH7.0, 150 mM NaCI, 1 mM EDTA, 1% NP-40, and a protease inhibitor cocktail (BMB) and resolved on a 10-20% SDS-PAGE gel. The bound ligand was identified by immunoblotting using anti-FLAG monoclonal antibody (BMB).

As shown in Figure 13A, the secreted FLAGhIL-1Ral fusion protein bound to IL-18R ECD and did not bind to IL-1R ECD, which indicates that hIL-1Ral could be an agonist or antagonist of IL-18R.

EXAMPLE IL-1 Receptor and IL-18 Receptor Binding of mIL-lRa3 and hIL-lRa3 cDNA encoding mIL-1Ra3 (DNA92505 shown in Figure 9; SEQ ID NO: 15) was cloned into pRK7 with a carboxy-terminal FLAG-tag. The resulting expression construct was transfected into human embryonic kidney 293 cells by means of calcium phosphate i 30 precipitation. 84-90 hours post transfection, the conditioned media containing secreted S* FLAGmIL-1Ra3 fusion protein was harvested. Conditioned media containing secreted IL- 18R-Fc and IL-1R-Fc proteins were prepared as described in Example 9 above, with the exception that the 293 cells were transfected with either pCMV1FLAG-IL1R-ECD-Fc or pCMV1FLAG-IL18R-ECD-Fc alone (without pCMV1FLAG-IL-lRal cotransfection).

35 For in vitro binding assays, IL-1R-Fc or IL-18R-Fc from 0.5 ml of the conditioned medium was immobilized to protein G-agarose and then mixed with 1.2 ml conditioned S-medium containing FLAGmIL-1Ra3. The receptor-ligand complexes were washed and resolved on an 10-20% SDS-PAGE gel and the bound ligand was detected by immunobloting using anti-FLAG monoclonal antibody (Boehringer Mannheim).

As shown in Figure 14, FLAGmIL-1Ra3 fusion protein bound to IL-1R ECD and did not bind to IL-18R ECD. Since the amino acid sequence of mIL-1Ra3 is related to that of the -58known interleukin-1 receptor antagonist protein (IL-1Ra), mIL-3Ra3 is believed to be a novel IL-1 receptor antagonist.

cDNA encoding hiL-lRa3 (DNA96787 shown in Figure 7; SEQ ID NO:12) was cloned into pRK7 with a carboxy-terminal FLAG tag to form pRK7hIL-1Ra3-FLAG. pCMV1FLAG- IL1R-ECD-Fc and pCMV1FLAG-IL18R-ECD-Fc were obtained as described in Example 9 above. Similarly, cDNA encoding DR6 was cloned into the modified pCMV1FLAG vector of Example 9 to form pCMV1FLAG-DR6-Fc, encoding DR6 fused to the Fc portion of human immunoglobulin G. Conditioned media containing a combination of secreted FLAGhIL- 1Ra3 and FLAG-DR6-Fc a combination of secreted FLAGhIL-1Ra3 and FLAG-IL1R-ECD- Fc or a combination of secreted FLAGhIL-1Ra3 and FLAG-IL18R-ECD-Fc were prepared by cotransfecting Human 293 cells with pRK7hIL-1Ra3-FLAG and pCMV1FLAG-DR6-Fc pRK7hIL-1Ra3-FLAG and pCMV1FLAG-IL1R-ECD-Fc or pRK7hIL-1Ra3-FLAG and pCMV1FLAG-IL18R-ECD-Fc, culturing the transfectant cells and harvesting culture media essentially as described in Example 9 above. The receptor-Fc and ligand complex from each conditioned medium was immunoprecipitated with protein G-Sepharose or anti-FLAG monoclonal antibody, and immunoprecipitates were resolved by gel electrophoresis and immunoblotting with anti-FLAG monoclonal antibody essentially as described in Example 9 above.

As shown in Figure 13B, FLAGhIL-1Ra3 fusion protein bound to IL-1R-ECD-Fc and did not bind to IL-18R-ECD-Fc or DR6-Fc. Since the amino acid sequence of hIL-1Ra3 is related to that of the known interleukin-1 receptor antagonist protein (IL-1Ra), hIL-3Ra3 is believed to be a novel IL-1 receptor antagonist.

EXAMPLE 11 Preparation of Antibodies that Bind IL-llp This example illustrates preparation of monoclonal antibodies which can specifically bind IL-llp.

Techniques for producing the monoclonal antibodies are known in the art and are S described, for instance, in Goding, supra. Immunogens that may be employed include purified IL-llp, fusion proteins containing IL-llp, and cells expressing recombinant IL-llp on 30 the cell surface. Selection of the immunogen can be made by the skilled artisan without undue experimentation.

Mice, such as Balb/c, are immunized with the IL-11p immunogen emulsified in complete Freund's adjuvant and injected subcutaneously or intraperitoneally in an amount from 1-100 micrograms. Alternatively, the immunogen is emulsified in MPL-TDM adjuvant (Ribi Immunochemical Research, Hamilton, MT) and injected into the animal's hind foot Spads. The immunized mice are then boosted 10 to 12 days later with additional immunogen S: emulsified in the selected adjuvant. Thereafter, for several weeks, the mice may also be S: boosted with additional immunization injections. Serum samples may be periodically obtained from the mice by retro-orbital bleeding for testing in ELISA assays to detect anti-ILllp antibodies.

-59- After a suitable antibody titer has been detected, the animals "positive" for antibodies can be injected with a final intravenous injection of IL-11p. Three to four days later, the mice are sacrificed and the spleen cells are harvested. The spleen cells are then fused (using polyethylene glycol) to a selected murine myeloma cell line such as P3X63AgU. 1, available from ATCC, No. CRL 1597. The fusions generate hybridoma cells which can then be plated in 96 well tissue culture plates containing HAT (hypoxanthine, aminopterin, and thymidine) medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells will be screened in an ELISA for reactivity against IL-11p.

Determination of "positive" hybridoma cells secreting the desired monoclonal antibodies against IL-llp is within the skill in the art.

The positive hybridoma cells can be injected intraperitoneally into syngeneic Balb/c mice to produce ascites containing the anti-IL-llp monoclonal antibodies. Alternatively, the hybridoma cells can be grown in tissue culture flasks or roller bottles. Purification of the monoclonal antibodies produced in the ascites can be accomplished using ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be employed.

EXAMPLE 12 Isolation of DNA encoding hIL-1RalL, hIL-1RalV and hIL-1RalS Several intron-containing cDNA clones related to the hIL-1Ral intron-containing clone DNA85066 (Figure 2) (SEQ ID NO:4) were isolated from a human testis cDNA library and fully sequenced. The intron-containing cDNA sequences were used to determine a fulllength open reading frame (ORF) with the GENESCAN program (http://CCR- 081.mit.edu/GENESCAN.html). The ORF-encoding sequence was used to design two DNA primers, ggc gga tcc aaa atg ggc tct gag gac tgg g (SEQ ID NO:29) (1Ral016) and gcg gaa ttc 25 taa tcg ctg acc tea ctg ggg (SEQ ID NO:30) (1Ra1017). The lRal016 and lRal017 primers S were synthesized and used to clone cDNA from human fetal skin and SK-lu-1 cell cDNA libraries using polymerase chain reaction (PCR). Several PCR products were isolated and sequenced. Two full length cDNA clones (designated DNA102043 and DNA102044) from PCR products were found to encode hIL-IRal isoforms.

30 The entire nucleotide sequence of clone DNA102043 is shown in Figure 15 (SEQ ID NO:18). Clone DNA102043 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 4-6, and a stop codon at nucleotide *positions 625-627 (Figure 15; SEQ ID NO:18). The predicted polypeptide precursor (designated hIL-1RalL) (Fig. 15; SEQ ID NO:19) is 207 amino acids long. The putative signal 35 sequence extends from amino acid positions 1-34.

Clone DNA102043 (designated DNA102043-2534) was deposited with ATCC and was assigned ATCC deposit no. 203846. The full-length hIL-lRalL protein shown in Figure o (SEQ ID NO:19) has an estimated molecular weight of about 23,000 daltons and a pi of about 6.08.

Based on a sequence alignment analysis of the full length sequence (SEQ ID NO:19), hIL-IRalL shows significant amino acid sequence identity to hIL-1RaP and TANGO-77 protein. In addition, a portion of the DNA sequence of clone DNA 102043 (Figure 15) (SEQ 1D NO:18) was found to coincide with the DNA sequence of EST AI014548 (Figure 4) (SEQ ID NO:8) and with the complement of the DNA sequence of EST AI323258 (Figure 17) (SEQ ID NO:23).

The entire nucleotide sequence of clone DNA102044 is shown in Figure 16 (SEQ ID Clone DNA102044 contains a single open reading frame with an apparent translational initiation site at nucleotide positions 4-6, and a stop codon at nucleotide positions 505-507 (Figure 16; SEQ ID NO:20). The predicted polypeptide (designated hIL- 1RalS) (Fig. 16; SEQ ID NO:21) is 167 amino acids long, and it is believed to behave as a mature sequence (without a presequence that is removed in post-translational processing) in certain animal cells. In addition, it is believed that other animal cells recognize and remove in post-translational processing one or more signal peptide(s) contained in the sequence extending from amino acid positions 1 to about 46.

Clone DNA102044 (designated DNA102044-2534) was deposited with ATCC and was assigned ATCC deposit no. 203855. The full-length hIL-1RalS protein shown in Figure 16 (SEQ ID NO:21) has an estimated molecular weight of about 18,478 daltons and a pi of about Based on a sequence alignment analysis of the full length sequence (SEQ ID NO:21), hIL-1RalS appears to be an allelic variant of TANGO-77 protein and also shows significant amino acid sequence identity to hIL-1Rap. In addition, a portion of the DNA sequence of clone DNA102044 (Figure 16) (SEQ ID NO:20) was found to coincide with the DNA sequence of EST AI014548 (Figure 4) (SEQ ID NO:8) and with the complement of the DNA sequence of EST AI323258 (Figure 17) (SEQ ID NO:23).

EST clone AI323258 was purchased from Research Genetics (Huntsville, AL) and the cDNA insert was obtained and sequenced in its entirety. The entire sequence of the clone AI323258, designated DNA114876, is shown in Figure 19 (SEQ ID NO:24). Clone DNA114876 contains a single open reading frame (ORF) with an apparent translation 30 initiation site at nucleotide positions 73-75 and a stop codon at nucleotide positions 726-728 (Figure 19; SEQ ID NO:24), encoding a predicted polypeptide precursor (hIL-1RalV) (Fig. 19; 0.09 SEQ ID NO:25) that is 218 amino acids long. In addition, the ORF contains an alternate translation initiation site at nucleotide positions 106-108. The predicted polypeptide (also designated hIL-1RalV) for translation initiated at the alternate start codon is 207 amino acids in length (lacking the first eleven residues at the N-terminus of the 218 amino acid polypeptide). It is believed that the predicted 218 amino acid and 207 amino acid polypeptides behave as mature sequences (without a presequence that is removed in post- Stranslational processing) in certain animal cells. It is also believed that other animal cells recognize and remove one or more signal peptide(s) extending from amino acid positions 1 to about 48 (a putative leader sequence in the 218 amino acid polypeptide) or from amino acid -61positions 12 to 36 (a putative leader sequence in the 207 amino acid polypeptide) in the amino acid sequence of Figure 19 (SEQ ID NO:25). As shown in Example 14 below, transiently transfected CHO host cells secrete unprocessed forms of hIL-iRaiV and hIL- IRalL and a single processed form that results from the removal of a signal peptide extending from amino acid positions 1 to 45 in Figure 19 (SEQ ID NO:25) or the removal of a signal peptide extending from amino acid positions 1 to 34 of Figure 15 (SEQ ID NO:19). The processed form of hIL-1RalV and hIL-1RalL secreted by transiently transfected CHO host cells has the amino acid sequence of amino acid residues 35 to 207 of Figure 15 (SEQ ID NO: 19) and amino acid residues 46 to 218 of Figure 19 (SEQ ID Clone DNA114876 (designated DNA114876-2534) was deposited with ATCC and was assigned ATCC deposit no. 203973. The full length hIL-1RalV protein shown in Figure 19 (SEQ ID NO:25) has an estimated molecular weight of about 24,124 and a pl of about 6.1.

Based on a sequence alignment analysis of the full length sequence (SEQ ID hIL-1RalV shows significant amino acid sequence identity to hIL-1Rap. hIL-1RalV is believed to be an allelic variant of hIL-1RalL.

EXAMPLE 13 IL-18 Receptor and IL-1Receptor Binding of hIL-1RalS To facilitate the characterization of hIL-1RalS, a PCR fragment encoding amino acid residues 39-167 in the ORF of clone DNA102044 (Figure 16; SEQ ID NO:21) was cloned into pCMV1FLAG (IBI Kodak, described in Pan et al., Science, 276: 111-113) as an in-frame fusion to a NH2-terminal preprotrypsin leader sequence and FLAG tag encoded by the vector to form plasmid pCMV1FLAG-IL-1RalS. Plasmid pCMV1FLAG-IL18R-ECD-Fc was obtained as described in Example 9 above.

Human embryonic kidney 293 cells were grown in high glucose DMEM (Genentech, Inc). The cells were seeded at 3-4 X10 6 per plate (100 mm) and co-transfected with pCMV1FLAG-hIL-lRalS and pCMV1FLAG-IL18R-ECD-Fc by means of calcium phosphate precipitation. The media were changed 12 hours post transfection. The resultant conditioned media (10 ml each) were harvested after a further 70-74 hour incubation, clarified by S: centrifugation, aliquoted and stored at -70C. The receptor-Fc and ligand complex from :30 ml conditioned medium was immunoprecipitated with protein G-Sepharose, washed three times with buffer containing 50 mM Hepes, pH7.0, 150 mM NaC1, 1 mM EDTA, 1% and a protease inhibitor cocktail (BMB) and resolved on a 10-20% SDS-PAGE gel. The bound ligand was identified by immunoblotting using anti-FLAG monoclonal antibody (BMB).

The immunoblotting results indicated that the secreted FLAGhIL-1RalS fusion *35 protein bound to IL-18R ECD. These data show that hIL-1RalS could be an agonist or antagonist of IL-18R.

EXAMPLE 14 hIL-1RalV. hIL-1RalL and hIL-lRa3 Processing cDNAs encoding full-length hIL-1RalV (amino acids 1-218 in the ORF of clone DNA114876 shown in Figure 19 (SEQ ID NO:25)), full length hIL-1RalL (amino acids 1-207 -62in the ORF of clone DNA102043 shown in Figure 15 (SEQ ID NO:19)), and full length hIL- 1Ra3 (amino acids 1-155 in the ORF of clone DNA96787 shown in Figure 7 (SEQ ID NO:13)) were each cloned into a pRK7 expression vector as an in-frame fusion with a carboxyterminal FLAG-tag sequence. In preparation for mammalian cell transient transfections, CHO DP12 cells were seeded at 4 x 106 cells per plate (100mm petri dish) in growth medium 5% FBS, IX GHT, IX pen/strep, 1X L-glutamine) the day before transfection. On the day of transfection, cells were washed with PBS and fed with 10 ml serum-free transfection medium (PS20, 1X GHT). DNA-lipid transfection mixtures were prepared by adding stepwise into eppendorf tubes 400 :1 transfection medium (PS20, IX GHT); 12 :g DNA; 10 :g poly-lysine; and 50 :1 Dosper liposomal transfection reagent (Boehringer Mannheim). The DNA-lipid mixtures were incubated for 15 minutes at room temperature and then added dropwise to cell culture plates. Cells were incubated overnight at 3 7 On the day after transfection, cells were washed with PBS, fed with 10 ml serum-free production medium (PS24, 10 mg/L insulin, IX trace elements, 1.4 mg/L lipid EtOH), and placed in a 32 0

C

incubator. After 5 days, the culture media containing the expressed proteins were harvested and cleared by centrifugation. For peptide sequencing of each expressed protein, 5-10 ml of the conditioned medium containing the expressed protein was incubated with monoclonal anti-FLAG antibody (Boehringer Mannheim) coupled to agarose beads. The immunoprecipitated FLAG-tag proteins were extensively washed with 1% NP-40 buffer (125 mM NaCI, 1 mM EDTA and 50 mM Tris-HC1, pH The immunoprecipitates were run on a SDS polyacrylamide gel, the separated polypeptides on the gel were transferred to a PVDF membrane, the PVDF membrane was stained with Coomassie blue, and the corresponding protein bands were excised from the membrane. The amino-terminal protein sequences were obtained by conventional methods.

25 The processed N-terminal sequence of both of the hIL-1RalL and hIL-1RalV S polypeptides was determined to be VHTSPKVKN (SEQ ID NO:31). Approximately 50% of hIL- IRalL and hIL-1RalV material recovered from conditioned media exhibited the processed Nterminal sequence, indicating that the CHO host cells secreted a processed form i corresponding to amino acid residues 35 to 207 in the amino acid sequence of Figure (SEQ ID NO: 19) and amino acid residues 46 to 218 in the amino acid sequence of Figure 19 (SEQ ID NO:25). The remaining 50% of the hIL-1RalL and hIL-1RalV material recovered from conditioned media exhibited an unprocessed N-terminus, indicating that the CHO host cells also secreted unprocessed forms of hIL-1RalL and hIL-1RalV corresponding to amino acid residues 1 to 207 in the amino acid sequence of Figure 15 (SEQ ID NO:19) and to amino 35 acid residues 1 to 218 in the amino acid sequence of Figure 19 (SEQ ID NO:25), respectively.

The processed N-terminal sequence of both of the hIL-1Ra3 and mIL-1Ra3 polypeptides was determined to be VLSGALCFRM (SEQ ID NO:33). Approximately 100% of the hIL-1Ra3 and mIL-1Ra3 material recovered from conditioned media exhibited the processed N-terminal sequence, indicating that the CHO host cells secreted processed forms of hIL-1Ra3 and mIL-1Ra3 that lack the N-terminal methionine and correspond to amino -63acid residues 2 to 155 in the amino acid sequence of Figure 7 (SEQ ID NO:13) and amino acid residues 2 to 155 in the amino acid sequence of Figure 9 (SEQ ID NO: 16), respectively.

Deposit of Material The following materials have been deposited with the American Type Culture Collection, 10801 University Blvd., Manassas, VA 20110-2209, USA (ATCC): Material ATCC Dep. No. Deposit Date pSPORT1-based plasmid 203586 Jan. 12,1999 DNA92929-2534 pCMV-1Flag-pcmv5 plasmid 203587 Jan. 12, 1999 DNA96786-2534 pT7T3D-Pac plasmid 203588 Jan. 12, 1999 DNA85066-2534 pINCY-based plasmid 203589 Jan. 12, 1999 DNA96787-2534 pT7T3D-Pac plasmid 203590 Jan. 12, 1999 DNA92505-2534 pRK7-based plasmid 203846 March 16, 1999 DNA102043-2534 pRK7-based plasmid 203855 March 16, 1999 DNA102044-2534 pRK7-based plasmid 203973 April 27, 1999 DNA114876-2534 These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty). This assures maintenance of viable cultures of the deposits for 30 years from the date of deposit. The deposits will be 35 made available by ATCC under the terms of the Budapest Treaty, and subject to an agreement between Genentech, Inc. and ATCC, which assures permanent and unrestricted S* availability of the progeny of the cultures of the deposits to the public upon issuance of the S* pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the J 45 materials will be promptly replaced on notification with another of the same. Availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

-64- The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the constructs deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and any constructs that are functionally equivalent are within the scope of this invention. The deposit of material herein does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

00 o *0 5° S S

S.

Table 2A

PRO

Comparison Protein

-X-XXXXXXXXXXXX

xxxxxYYYYYYY (Length 15 amino acids) (Length 12 amino acids) amino acid sequence identity (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) divided by 15 33.3% Table 2B

PRO

Comparison Protein

XXXXXXXXXX

XXXXXYYYYYYZZYZ

(Length 10 amino acids) (Length 15 amino acids) amino acid sequence identity (the number of identically matching amino acid residues between the two polypeptide sequences as determined by ALIGN-2) divided by (the total number of amino acid residues of the PRO polypeptide) divided by 10 Table 2C

NNNNNNNNNNNNNN

NNNNNNLLLLLLLLLL

PRO-DNA

Comparison DNA (Length 14 nucleotides) (Length 16 nucleotides) nucleic acid sequence identity (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) 35 6 divided by 14 42.9% 9 9 9 9 .9 9 9 @0 o 9 0*e 9 9 9*9* Table 2D

PRO-DNA

Comparison DNA

NNNNNNNNNNNN

NNNNLLLVV

(Length 12 nucleotides) (Length 9 nucleotides) nucleic acid sequence identity (the number of identically matching nucleotides between the two nucleic acid sequences as determined by ALIGN-2) divided by (the total number of nucleotides of the PRO-DNA nucleic acid sequence) 4 divided by 12 33.3% -66- Table 3A C-C increased from 12 to Z is average of EQ B is average of ND match with stop is stop-stop 0; J (joker) match 0 #define _M -8 value of a match with a stop int -day[26] [26] =I *A 2, 00,-41-1-0-1,-2,-1,0,_MI1 10,0,60,30, I* BI 10, 3,4, 0,l1,-2, 0, 0, 1 c 0, *D 103,-54,3,-61,1-2,0,0-4-3,2,_M-1 2-1,0,0,0-2,-7,04 E* 10, 3, 0,I1,-2, 0, 1,_M,-l1 0,0, 3}, F 2, 0,0, G 1, 0), I* H *1 0, 0, 3, 0, 0, 2}, 1 5, 2, 0, 1 0,0, 0, 0,0,0, 0,0, 0,0, 0, 0, 0,0,0, 0, 0,0, 0, 0, 0, 0), 3, 0), 2, 6, 0,2,0,,2) I* M 1- 2, 0, N 0,2,42, 1, 0,1,0,0-2,40,21, 1* 0* LmM, M-M-M_-M-M_-M-M-M-M-MMM MM M M-MM-MM -MM,M}l P 6, 0, 0, 1, 0, 0,-1,60,50) Q 2, 0, 1, 0, 0,4, 3), I* R *1 0, 00_OM, 0, 1, 2, 0,4, 0), 1, 0,0, 00,-31 1,-102,1,0-1,20,30) I* 1, 0, 0, 1, 3, 0), 0,0, 0,0, 0, 0,0, 0,0, 0, 0,0_M, 00,0,0,0,0,0,0, 0,0, 0), 2, 00,4,60,2-) W *1 0,-6,17, 0, 0,-20,,04, 0, 1,-52,3-5, 02,-200,-2-Il _M0,3, 00,0,0-2,-,,44

PS-S

*00S 4*S 0

S.

0 45 54 .4 0 0*00

S.

~0 050 0

I

-67- Table 3B #include <stdio.h> #include <cty-pe.h> #define #define #define #define #define #define #define #define #define #define MAXJM P

MAXGAP

JMPS

MX

16 24 1024 4

DMAT

DMIS

DINSO

DINS I

PINSO

P INS I I* max jumps in a diag don't continue to penalize gaps larger than this I* max jmps in an path *I I* save if there's at least MX-lI bases since last jmp 1* value of matching bases *I 1* penalty for mismatched bases penalty for a gap I* penalty per base I* penalty for a gap 1* penalty per residue structjmp I short unsigned short struct diag I int long short structjmp n[MAXJMP; x[MAXJMPI; size of jmp (neg for dely) 1* base no. ofjmp in seq x I* limits seq to 2AI6 -1 score; offset; ijmp;I 1* score at last jmp offset of prey block 1* current jmp index 1* list ofjmps *1 1* number of leading spaces struct pathI int short n[JMPS];I* size ofjmp (gap) int x[JMPS]; loe ofjmp (last elem before gap) char char char char int int int int int long struct struct *namex[2]; *prog; *seqx[2]; dmax; dmax0; dna; endgaps; gapx, gapy; lenO, len]; ngapx, ngapy;I smax; *xbm; offset; *dx; I* output file name seq names: getseqs()O prog name for err msgs seqs: getseqs()o best diag: nwo)* 1* final diag 1* set if dna: maino 1* set if penalizing end gaps total gaps in seqs seq lens total size of gaps 1* max score: nw0 o bitmap for matching 1* current offset in jmp file holds diagonals holds path for seqs 55 char char *calloco, *mal loco, *indexo, *strcpyo; *getseqo, *gcalloco; -68- Table 3C Needleman-Wunsch alignment program usage: progs file I file2 where file I and file2 are two dna or two protein sequences.

The sequences can be in upper- or lower-case an may contain ambiguity Any lines beginning with are ignored Max file length is 65535 (limited by unsigned short x in the jmp struct) A sequence with 1/3 or more of its elements ACGTU is assumed to be DNA Output is in the file "align.out" The program may create a tmp file in Atmp to hold info about traceback.

Original version developed under BSD 4.3 on a vax 8650 #include "nw.h" #include "day.h" static -dbval[26] 1, 14,2,13,0,0,4,11,0,0, 12,0,3, 15 ,0,0,0,5 ,6,8,8,7,9,0,10,0 1; static _pbval[26]= 1, 21( 4, 8, 16, 32, 64, 128, 256, OxFFFFFFF, 1<<10, 1-l1, 1-12, 1l«13, 1-14, 1l-15, 1l-16, 1I<<17, 1l-18, 1I<<19, 1l-20, 1-2 1, 1<'-22, 1-23, 1<<24, main(ac, av) main int ac; char prog =av[0I; if (ac 3) fpri ntf(stderr, "usage: %s filel file2\n", prog); fprintf(stderr,"where file I and filc2 are two dna or two protein sequences.\n"); fprintf(stderr,"Thie sequences can be in upper- or lower-case\n'); fprintf(stderr,Any lines beginning with are ignored\n"); fprintf(stderr,"Output is in the file \'align.out\"); exit(l); :namex[ I] av[2]; seqx[0] getseq(namex[0], &lenO); .045 seqx[ I] =getseq(namex[lI], &len 1); xbm (dna)? _dbval pbal :endgaps 0; I to penalize endgaps ofile ="alignout"; I* output file 50.

nwo~; fill in the matrix, get the possible jmps readjmpso; get the actual jmps printo; I* print stats, alignment cleanup(0); unlink any tmp files -69- Table 3D do the alignment, return best score: maino dna: values in Fitch and Smith, PNAS, 80, 1382- 1386, 1983 *pro: PAM 250 values When scores are equal, we prefer mismatches to any gap, prefer a new gap to extending an ongoing gap, and prefer a gap in seqx to a gap in seq y.

nw() fw char *px, *py; I* seqs and ptrs int *ndely, *dely; keep track of dely int ndelx, dclx; keep track of dclx int *tmp; for swapping rowO,.row 1 5 int mis; score for each type int insO, ins I; insertion penalties register id; diagonal index register ij;' /*jmp index register *colO, *col 1; score for curr, last row register xx, Yy; index into scqs dx =(struct diag calloc("to get diags", lenO+len 1 sizeof(struct diag)); ndely (int calloc('to get ndely', len 1 1, sizeof(int)); dely (int calloc("to get dely', lenl1+l, sizeof(int)); colO (int calloc('to get colO', lenl1+l, sizeof(int)); coil (ilt calloc("to get coil len 1+1, sizeof(int)); insO (dna)? DINSO PINSO; ins I (dna)? DINS I PINS 1; smax =-10000; if (cndgaps) I for (co]0[0] dely[0] -insO, yy 1; yy len I; yy++) colO[yy] dely[yy] colO[yy-1] ins I; ndely[yy] yy;colO[0] Waterman Bull Math Biol 84 else for (yy yy len1; yy++) dely[yy) -insO; 1* fill in match matrix .*45 for (px =seqx[0], xx I xx lenO; px+4, xx++) 1* initialize first entry in col if (endgaps)f :so if (xx 1) :5els col delx =-(ins0+ins); col 1[0] delx colO[O] insl1; ndelx =xx; else I :col 1[0] 0; delx 0is; ndelx 0 is; Table 3E for (py= seqx[l], yy= 1; yy<= len I; yy++) mis COlO[yy- I]; if (dna) mis (xbmr[*px-'A']&xbni-'A) DA: MS else mis _day[ I* update penalty for del in x seq; favor new del over ongong del ignore MAXGAP if weighting endgaps if (endgaps 11 ndely[yy] MAXGAP) if (colO[yy] insO >=dely[yy]) I dely[yy] =colO[yy] (insO+insl); ndely[yy] =1 elseI dely[yy] insi; ndely[yy]++;, Ielse if (colO[yy] (insO+insl) dely[yy]) I dely[yyj colO[yy) (insO+insl); ndely[yy] 1; else ndely[yy]++;- 1* update penalty for del in y scq;- *favor new del over ongong del if (endgaps 11 ndelx MAXGAP) I if (col I[yy- I]I insO delx){ delx =col I[yy-lI] (insO+insl); ndelx =I; elseI delx ins]; ndelx++; else if (collI[yy-l] (insO+insl) deix){ delx =coll[yy-]] -(insO+insl); *ndelx I; le ndelx++; pick the maximum score; we're favoring :50 :mis over any del and delx over dely Table 3F 0:::o.55 id=xx-yy+lenl -1; if (mis delx mis dcly[yy]) ele collI[yy] mis, esif(delx dely[yy]) I col I[yyl delx; iidX[id].ijr-p; dx i]jp.n[O] (!dna 11 (ndelx >MAXJMP xx dx[id].jp.xfijj+MX) 11mis dxfid).score±VINSU)){ dx~id].ijmp--t; if (-Hij MAXJMP){ writejmps(id); ij dx[id].ijmp 0;1 dx[id].offset =offset; -71offset sizeof(struct jmp) sizeof(offset);, dx[id].jp.nf i J] ndelx:.

dx[id].jp.x[ij] xx; dx[id].score deix; elseI col I yy] dely[yy]; ij dx[id].ijmp; if (dx[id].jp.n[O] (!dna 11 (ndely[yy] MAXJMP xx dx[id].jp.x[ij]+MX) 11 mis dx[id].score+DINSO)){ dx[id].ijmp++; if MAXJMP)I writejmps(id); ij dx[id].ijmp 0; dx[idl.offset offset; offset sizeof(structjmp) sizeof(offset); dx[id].jp.n~ijI -ndely[yy]; dx[id].jp.x[ij] xx; dx[idl.score dely[yy]; if (xx =lenO yy len 1)1 last col if (endgaps) collI[yy] ins0Osins *(len I yy); if (colI I[yyj smax) I smax collI yy]; dmax =id; if (endgaps xx lenO) col I[yy- I] ins0+ins I*(len0-xx); if (col I[yy- I smax) smax col I[yy- I] dmax id; ::tmp colO; colO =col1; col I tmnp; (void) free((char *)ndely); (void) frcc((char *)dely); (void) free((char *)colO); (void) free((char *)colI1); -72- Table 3G *print() only routine visible outside this module *Static: *getmat() trace back best path, count matches: printo *pr align() print alignment of described in array printo *dumpblocko dump a block of lines with numbers, stars: pralign() numso put out a number line: dlumpblocko putlineo put out a line (name, [num], seq, dumpblocko starso -put a line of stars: dumpblock() stripnameo strip any path and prefix from a seqname #include 'nw.h" #define SPC 3 #define P_-LINE 256 maximum output line #define P_SPC 3 space between name or num and seq extern _day[26][26]; int olen; set output line length FILE *fx; output file printo print int Ix, ly, firstgap, lastgap; 1* overlap if ((fx fopen(ofile, 0)1 fprintf(stderr,"%s: can't write prog, ofile); cleanup( I); }pit~x <is eune s(egh=%)nnmxOln) ~fprintf(fx, "<first sequence: %s (length namx[], len olen lx lenO; ly lenI1; firstgap lastgap =0; if (dmax len I I) I leading gap in x pp[0].spc =firstgap len I dmax 1; ly pp[0].spc; *else if (dmax len I 1* leading gap in y pp[lI].spc firstgap dmax (len I 1); lx pp[ I I.spc; 50if (dmax0 lenO I) trailing gap in x *lastgap =lenO dmax0 1; 50 ls x lastgap; eleif (dmax0 lenO 1) trailing gap in y lastgap =dmax0 (lenO 1); ly lastgap; getmat(lx, ly, firstgap, lastgap); pr*lino -73- Table 3H *trace back the best path, count matches static getmat(lx, ly, firstgap, lastgap) getinnat int lx, ly;- "core" (minus endgaps) int firstgap, lastgap; leading trailing overlap int nm, iO,i 1, siz0, siz I; char outxf 321; double pct; register nO, n 1; register char *p0, *pI; get total matches, score iO il siz sizi 0; p0 seqx[0] pp[ I].spc; p I seqx[I]+ pp[0].spc; n0=pp[lI].spc I; n I =pp[O].spc I; nm =0;1 while( *p0 *pI if (siz0) I n I sizO--;else if (siz 1) pO++.

siz I else if (xbm[ *pO0'A'] &xbm[ *plI n m-H-; if -pp[0].x[iO]) sizO if (n ==pp[lI].x[ilI]) P+ siz I pp[ I].n[i II+]; 50 pct homology: *if penalizing endgaps, base is the shorter seq 50 ~else, knock off overhangs and take shorter core if (endgaps) Ix (lenO fenl1)? lenO len 1; else 55 Ix (lX<ly)? Ix :ly; pct 100.*(double)nnmd(double)lx; fprintf(fx, fprintf(fx, 0 0 d match%s in an overlap of 0 /od: %.2f percent similarity\n", 0nm, (nm es", lx, pct); -74- Table 31 fprintffx, "<gaps in first sequence: gapx), if (gapx) I (void) sprintf(outx, ngapx, (dna)? "base"';residue", (ngapx fprintf(fx,"%s", outx); fprintf~fx, gaps in second sequence: gapy); if (gapy) I (void) sprintf(outx, ngapy, (dna)? "base": "residue", (ngapy I fprintf(fx,"%s", outx); getmat if (dna) else fprintf(fx, "\n<score: %d (match mismatch gap penalty O/d %d per base)\n", smax, DMAT, DMIS, DINSO, DINS I); fpri ntf(fx, "\n<score: %d (Dayhoff PAM 250 matrix, gap penalty %d per residue)\n", smax, PINSO, PINS I); if (endgaps) fprintf(fx, "<cndgaps penalized, left endgap: %d right endgap: %d firstgap, (dna)? "base" "residue", (firstgap I "s" lastgap, (dna)? "base" "residue", (lastgap else fprintf(fx, "<endgaps not penalized\n"); static static static static static static static char static char static char static char nm; Imax; ij[2]; nc[2]; ni[2]; siz[2]; *ps[2]; out[2][PLINE]; star[P LINE]; matches in core for checking lengths of stripped file names jmp index for a path number at start of current line current elemn number for gapping 1* ptr to current element *I ptr to next output char slot output line set by starso a.

S S 9 *0 9 *b 0*

S

S 6 :50

C,

9 9 *v.9 9 55 a a ~59S 9 9.

S.

99

S

996 S 90 *print alignment of dcscribed in struct path pp[] static pral ign()

I

pralign nn; char count more; int register for (i 0,lImax 0; i I nn stripname(namcx[i]); if (nn Imax) Imax nn; nc[i]= 1; ni[i] 1; siz[i]j un] 0; ps[i] =seqx[i]; nnfil itfil Table MJ for (nn =nm 0, moure 1; more; pr_align for (i =more i 2; *do we have more of this sequence? if continue; more++; if (PP[iJ.spc) I I' leading space ppli].spc--; else if (siz[iI) I in a gap PONi14 else we're putting a seq element *po[i] *ps[i]; if (islower(*ps[iI)) *ps[i] toupper(*ps[i]); poliI++; Isll+ *are we at next gap for this seq? if (ni[i] we need to merge all gaps at this location siz[i] while (ni[i] pp[i].x[ij[i]]) ni[iJ++; 945if =olen 11 !more nn) 145 dumphlocko; for (i 0; i 2; 0 nn= 0;po[i] out[i]; nn *dump a block of lines, including numbers, stars: pr aligno 55 static dumpblock() dumphiock register i (i i -76- Table 3K dumpblock (void) putc('\n', fx); for i if (*out[iJ (South]j 11 )I if (i 0) nums(i); if (i 0 *out[lI]) starso; putline(i); if (i 0 *out[ I]) fprintf(fx, star); if I) nums(i);l 1 *put out a number line: dumpblocko static nums(ix) nums 5**55 61) int ix; I* index in out[] holding seq line char nline[PLINE]; register j register char *pn, 5 px, 5 py; for (pn =nline, i i Imax+PSPC; pn++) *pn for (i nc[ix], py =out[ixl; *py; py-H-, pn++) if (*Py IIpy*') elseI if (i%l10O 0 11 (i I nc[ixl 1 j -i i for (px pn; j;j 10, px--) *px =j%l0 if (i 0) px *pn pn nc[ix] i for (pn nline; *pn; pn++) (void) putc(*pn, fx); (void) putc('\n', fx); *put out a line (name, [num], seq, dumpblocko static int ix; Dutline -77- Table 3L putline int i.

register char *px; for (px namex[ix], i *px *p>x (void) putc(*px, fx); for I i< lmax+PSPG; i-I-I) (void) putcQ, fx); 1* these count from 1: ni[] is current element (from 1) nc[] is number at start of current line for (px =out[ix]; *px; (void) putc(*px&0x7F, fx); (void) putc(\n', fx); *put a line of stars (seqs always in out[O], out[lI]): dumpblock() static starso stars int i.

register char *po, *pl, cx, *pX; if 11 (*out[0I )I !*out[lI] I1 (*out[ I I retu rn; px star; for (i lmax+P_SPC; i; for (p0 out[0], p1 out[1]; *pQ *pl; pl++) if (isalpha(*p0) isalpha( if (xbm[*pO'A']&xbm[*p1'Ai]){ cx else if (dna 0) else cx else cxcx; 55 *p ='O -78- Table 3M *strip path or prefix from pn, return len: pralign() static siripname(pn) stripilame, char *pfl; file name (may be path)

I

register char ~px, py; py =0; for (px =pn; *px; px++) if (*px 'I) py =px 1; if (py) (void) strcpy(pn, py); return(strlen(pn)); Table 3N cleanupo cleanup any imp file getseq() read in seq, set dna, len, maxlen g calloc()- calloco with error checkin readjmpso -get the good imps, from tmp file if necessary writejmps() write a filled array ofjmps to a tmp file: nw() #include "nw.h" #include <syslfile.h> char *jname ="Itmp/homgXXXXXX'; tmp file for jmps FILE fi int cleanupo; cleanup tmp file long Iseeko; *remove any inp file if we blow :.cleanup(i) it i; cleanup if (fj) (void) unlinkojname); read, return ptr to seq, set dna, len, inaxlen skip lines starting with or'>* seq in upper or lower case char* getseq(file, len) getseq char *file; 1* file name t *len; 1* seq len :char line[ 1024], *pseq; register char *px, *py; int natgc, tlen; FILE *fp.

if((fp fopen(file,"r")) fprintf(stderr,"%s: can't read prog, file); exit( I); -79tien natgc 0; while (fgets(line, 1024, fp))I if (*line 11 flin fIline continue; for (px line; *px px++) if (isupper(*px) 11islower( *px)) tlen++; if ((pseq =malloc((unsigned)(tlen+6))) 0) 1 fprintf(stderr,"%s: malloco failed to get %d bytes for prog, tlen+6, file); exit( 1); pseq[0] pseq[ I] pseq[2] pseq[3] Table py pseq 4; gte *len tien; rewind(fp); while (fgets(line, 1024, fp)) if (*line I 11 *hne flncontinue; for (px line; *px px++) if (isupper(*px)) py++ *px; else if (islower(*px)) py++ toupper(*px); if (index("ATGGU",*(py-l))) natgc'-'; *py (void) fcloseffp); dna natgc (tlenI3); return(pseq+4); char gcalloc(msg, nx, sz) gcalloc .char *msg; program, calling routine mnt nx, sz;1 number and size of elements char *px, *calloco; **if ((px =calloc((unsigned)nx, (unsigned)sz)) 0) I fprintf(stderr, g_calloc() failed %s (n= 0 /od, prog, msg, nx, sz); ext~l) retu rn(px); get final jmps from dx[] or trnp file, set reset dmax: maino readjmpso readj mps t fd int siz, 10, i I; register i,j, xx; if 1 (void) fclose(j); if (fd open(.name, 0 RDONLY, 0) fprintfqstderr, can't open() prog, jnamc); cleanup( I); for (i iO i I 0, dmaxO =dmax, xxleIn0; while for 0 dx[dmax].ijmp; j 0 dx[dmax].jp.xUj] xx; Table 3P readjmps if j 0 dx[dmax].offset fj) (void) lseek(fd, dx[dmax].offset, (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct imp)); (void) read(fd, (char [dmax]. offset, sizeof(dx [d max]. offset));dx[dmax].ijmp MAXJMP-I; else break;, if (i >=JMPS){ fprintf(stderr, too many gaps in alignment\n', prog); cleanup( I); if 0 siz dx[dmax].jp.nUj]; xx dx[dmax].jp.xoj]; dmax six; if (Siz 0) gap in second seq pp[ I].n[i I] -six;xx six; 1* id xx yy lenl pp[I].x[iI] xx dmax leni 1; gapy-H-; ngapy siz; I* ignore MAXGAP when doing endgaps six (-six MAXGAP 11endgaps)? -six: MAXGAP; I else if (six 0) I* gap in first seq pp[0].n[iO] six; :45pp[0].x[iO] xx; gapx++; :ngapx six; ignore MAXGAP when doing endgaps *I 50six (six MAXGAP 11 endgaps)? six: MAXGAP;

I

else break; 551 reverse the order ofjmps ***for j=0, j <iO; jll, i0--){I i pp[0].njj]; pp[0].nUj] pp[0].n[iO]; pp[0].n[iO] i i pp[Oj-xWj; ppfO].xb] pp[0].x[iO]; pp[0].xiO] i C fordf ii. ;cIi4+ il II i p[l pp[l =pp[I =i- ~i =pp[l].xuj];pp[I].xuj]= pp[I].x[iI]; pp[I].xliI] =i if (fd 0) (void) closc(fd); if (void) unlinkojnamc); fj 0; offset 0; T able *write a filled jmp struct offset of the prey one (if any): nwo writejmps(ix) writej mps int ix; char *mktempo; if(!OD) if (mktempojname) 0) fprintfqstderr, can't mktempo prog, jaine); cleanup( I); if ((fj fopenojname, 0) j fprintftstderr, can't write prog, jaine); exit( I); (void) fwrite((char *)&dx[ix]jp, sizeof(struct jmp), 1, (void) fwrite((char *)&dx[ix].offset, sizeof(dxi xl].offset), 1, fj);- -82- EDITORIAL NOTE APPLICATION NUMBER 25935/00 The following Sequence Listing pages 1 to 19 are part of the description. The claims pages follow on pages "83" to "88".

Sequence Listing 0 0 0000.

0 0.

0 <110> Cenen <120> IL-i <130> P2534 <140> PCT/U <141> 1999- <150> US 60 <151> 1998- <150> US 60 <151> 1999- <150> US 60 <151> 1999- <160> 32 <210> 1 <211> 1006 <212> DNA <213> Homo <400> 1 ggcacgaggc agctactgcc tgatgttact cagcacctta gggt caagga ctgcatgtga agaaaccaaa cattcttgac caagtgcctg aagaaat tca tgggaatctc tctttgcatt ccgattctcc ggat aaagga tgaaactcac agggctcagg tech Inc. et al.

Related Polypept ides -3 S99 /3 0720 12-22 /113,430 12-23 /116, 843 01-22 /129, 122 04-13 sapiens aagccttcca ctacagaaag gctgctgttg agaccactca t catgagcga gacgctgaga ggaaagaaca agtcactggc agctctttgc gcattcatga atagcagttc agcctcatcc tgggggtctc caaagtcatc tcacccaaaaq tgggctcctg ggttatcgtg ttactagtgc gagtacaact caccttcaga gaacaccact tcctatgtca gctttaagaa ccagcctggg agaggtccaa ccaggatcac cagataaaaa ttgagctcag t aaaggg gag catcccttca qaatcagcac gaacatgctg acgcaccttg cctaaagctg tccctataga gtggccttga taagaggata ggctgtgata gcgcttaaga ggcccctgtt aggtgaagaa aaagtactgg ctacatacgc cctctgcgga ttttgtctct gctgaagaag qccggccctt gagtcggcgg aaagtctgag gcgctggcac aaacaactgc gaaagatttg gtgaactagt ggagggaaac gccacccacc ctttatcaaa cttaaacccg tcctggactc ccagagatct gaaaggaagt actgtgacaa gagaaactga catcttttat ctcaccccgg 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 atggttcatc tgcacctcct gcaattgtaa tgagcctgtt ggggtgacag 850 ataaatttga gaacaggaaa cacattgaat tttcatttca accagtttgc 900 aaagctgaaa tgagccccag tgaggtcagc gattaggaaa ctgccccatt 950 gaacgccttc ctcgctaatt tgaactaatt gtataaaaac accaaacctg 1000 ctcact 1006 <210> 2 <211> 26 <212> PRT <213> Homo sapiens <400> 2 Met Leu Leu Leu Leu Leu Giu Tyr Asn Phe Pro Ile Giu Asn Asn 1 5 10 Cys Gin His Leu Lys Thr Thr His Thr Phe Arg <210> 3 <211> 167 <212> PRT <213> Homo sapiens <400> 3 Val Lys Asn Leu Asn Pro Lys Lys Phe Ser Ile His Asp Gin His Asp Ser Gly Gly Lys Tyr His Val Lys Lys Leu Val1 Gin Leu Arg Pro Gly Val Asn Ser Ser Ser Ala Ala Gly Val1 Leu Val Tyr Ile Ser Ala Lys Gly His Pro Ala Gin Gin Val Trp Phe 125 Thr Asp 140 10 Leu Arg Ser Giu Ser Lys Gly Ile Lys Asp Pro Ala Phe Leu Giu Ser cy 5 Phe Ser Glu Glu Cys Gin Ser Trp Thr Glu Gly Ile Lys Leu Leu Aia Asn Ser Asn Asn 25 Phe 40 Gly 55 Tyr 70 Lys 85 Arg 100 Met 'is Cys 130 Arg Leu Phe Ser Cys Lys Arg Leu Asn Lys Ile Ala Pro Asp Giu Pro Giu Cys His Ala Leu Ile Lys Lys Phe Ser Asn Ile Val1 Ala Leu Asp Leu Ile Ala Giu Giu Asp Pro Ser Leu Lys Met Phe 105 Ala 120 Pro 135 Phe Ser Phe Gin Pro Vai Cys Lys Ala Giu Met Ser Pro Ser Giu Val Ser Asp 160 <210> 4 <211> 650 <212> DNA <213> Artificial Sequence <220> <223> recombinant DNA <400> 4 taattcacca tgtctgcact tgctgactac aaagacgatg tttgcagagg tccaaaggtg catgaccagg atcacaaagt agttccagat aaaaactaca catccttgag ctcagcctct gtctctaaag gggagttttg tcatccatcc cttcagctga aaaaggaatc agcacgccgg tcctggaaca tgctggagtc ctcctgcaat tgtaatgagc ggaaacacat tgaattttca cccagtgagg tcagcgatta <210> tctgatccta acgacaagct aagaacttaa actggtcctg tacgcccaga gcggagaaag tctctactgt agaaggagaa cccttcatct ggcggctcac ctgttggggt tttcaaccag gggtaccagt gctcttgttg tgcggccgcg acccgaagaa gactctggga gat ct tc tt t gaagtccgat gacaaggata actgatgaag tttatagggc cccggatggt gacagataaa tttgcaaagc cgactctaga gagctgcagt aattcagctc attcagcatt atctcatagc gcattagcct tctcctgggg aaggacaaag ctggctgccc tcaggtgggc tcatctgcac tttgagaaca tgaaatgagc ggatcccggg s0 100 150 200 250 300 350 400 450 500 550 600 650 Ala Ala Phe C ly Ile <211> 203 <212> PRT <213> Artificial Sequence <220> <223> recombinant protein <400> Met Ser Ala Leu Leu Ile Leu 1 5 Asp Tyr Lys Asp Asp Asp Asp Leu Cys Arg Gly Pro Lys Val Ser ie His Asp Gln Asp His Asn Leu Ile Ala Val Pro Asp Ala Leu Val 10 Lys Leu Ala 25 Lys Asn Leu 40 55 Lys Asn Tyr 70 Gly Ala Ala Val Ala Ala Asn Ser Asn Pro Lys Lys u Ap S or Ile Arg Pro Glu Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Giu Lys Gly Ser Pro Ile Tyr Cys Asp Lys Lys Lys Glu Lys Arg Arg Pro Phe Met Leu Glu Ser Cys Asn Cys Asn Arg Lys His Ile Met Ser Pro Ser Leu Asp 110 Leu 125 Ile 140 Ala 155 Glu 170 Giu 185 Giu 200 Leu Gly Val Ser Lys Gly Gin Ser Met Lys Leu Ala Phe Tyr Arg Ala Ala His Pro Gly Pro Val Gly Val Lys 100 His 115 Ala 130 Gin 145 Trp 160 Thr 175 Gly Glu Phe Cys Pro Ser Leu Gin Gin Lys Glu Ser Val Gly Ser Trp Phe Ile Cys Thr Asp Lys Phe Glu Leu 105 Leu 120 Ala 135 Asn 150 Ser 165 Asn 180 Phe Ser Phe Gin Pro Val Cys Lys Ala Glu 190 195 Vai Ser Asp <210> 6 <211> 754 <212> DNA <213> Homno sapiens ggcacgaggc agctactgcc tgatgttact cagcacctta gaaattcagc ggaatctcat tttgcattag gattctcctg ataaaggaca aagctggctg ggctcaggtg ggttCatctg aaat ttgaga aagccttcca ggttatcgtg acgcaccttg aaagtctgag ctacagaaag gctgctgttg agaccactca attcatgacc agcagttcca cctcatcctt ggggtctcta aagtcatcca cccaaaagga ggctcctgga cacc-ctgc acaggaaaca ttactagtgc gagtacaact caccttcaga aggatcacaa gataaaaact gagctcagcc aaggggagt t tcccttcagc atcagcacgc acatgctgga cattgaattt cctaaagctg tccctataga gtgaagaact agtactggtc acatacgccc tctgcggaga ttgtctctac tgaagaagga cggcccttca gtcggcggct agcctgttgg tcatttcaac gcgctggcac aaacaactgc taaacccgaa ctggactctg agagatcttc aaggaagtcc tgtgacaagg gaaactgatg tcttttatag caccccggat ggtgacagat cagtttgcaa 100 150 200 250 300 350 400 450 500 550 v %J 650 agctgaaatg agccccagtg aggtcagcga ttaggaaact gccccattga 700 acgccttcct cgctaatttg aactaattgt ataaaaacac caaacctgct 750 cact 754 <210> 7 <211> 193 <212> PRT <213> Homo sapiens <400> 7 Met Leu Leu Leu Leu Leu Giu Tyr Asn Phe Pro Ile Glu Asn Asn 1 Cys Asn Vai Ile Ala Gly Pro Gin Val1 Phe Asp Gin His Pro Lys Leu Asp Arg Pro Ser Aia Giu Phe Ser Leu Lys Giu Gly Ser Ile Cys Lys Phe Leu Lys Ser Giu Giu Cys Gin Ser Trp Thr Glu Lys Thr Phe Ser Giy Asn 50 Ile Phe Lys Gly 80 Leu Tyr Leu Lys 110 Ala Arg i2 5 Asn Met 140 Ser Cys 155 Asn Arg 170 Thr Ile Leu Phe Ser Cys Lys Arg Leu Asn Lys His His Ile Ala Pro Asp Giu Pro Giu Cys His Thr Phe 25 Asp Gin 40 Ala Val 55 Leu Ala 70 Ile Leu 85 Lys Asp 100 Lys Leu 115 Phe Ile 130 Ser Ala 145 Asn Glu 160 Ile Glu 175 Arg Asp Pro Ser Leu Lys Met Phe Ala Pro Phe Val1 His Asp Ser Gly Gly Lys Tyr His Val Ser Lys Lys Lys Leu Val1 Gin Leu Arg Pro Gly Phe Asn Leu Val Leu Asn Tyr Ser Ser Ser Lys Ser His 105 Ala Ala 120 Ala Gin 135 Gly Trp Val Thr 165 Gin Pro 180 Vai Cys Lys Ala Giu Met Ser Pro Ser Giu Val Ser Asp 185 190 <210> 8 <2i1> 629 <212> DNA <213> Hom'o sapiens <220> <221> unsure <222> 13 <223> unknown base <400> 8 ccaggcccaa gcntccccac catgaatttt gttcacacaa gtcgaaaggt 0* 0 0* 0 0 0 0 0* 00 00 0 0* 00 0000 0**0 0 0 0* 000000 gaagagctta. aacccg tactggcctg gactct tacgcccaga gatctt gcggagaaag gaagtc tctctactgt gacaag agaaggagaa actgat cccttcatct tttata ggcggctcac cccgga ctgttggggt gacaga tttcaaccag tttgca ggaaactgcc ccattg aaaaccccaa acctgc <210> 9 <211> 1321 <212> DNA <213> Homo sapiens <400> 9 gtcgacccac gcgtcc tgtagataaa gaccct aggcactcca ggagac tcactgttgc tgttat agaggggatc ccattt ttgtgagaag gtitgga tcatggatct gtatgg cgtgccaaga ctggta ctggttcatt gcctcc aacttgggaa gtcata actcagccta gaggtg caatqtgttt tcgtct agacaggagc aaggct caattacttc atagca agctgggtgg tataag aaga ggga cttt cgat gata gaag gggc tggt t aaa aag c aacg aattcagcat atctcatagc gcattagcct t c tcc tgggg aaggacaaag ctggctgccc tcaggtgggc tcatctgcac tttgagaaca tgaaatgagc tcatgaccag agttccagat cat cc ttgag gtctctaaag tcatccatcc aaaaggaatc tcctggaaca ctcctgcaat ggaaacacat cccagtgagg gatcacaaag aaaaactaca.

ctcagcctct gggagttttg cttcagctga agcacgccgg tgctggagtc tgtaatgagc tgaattttca tcagcgat ta ccttcctcgc taatttgaac taattgtata 600 tcac taaaaaaaa 629 gaag ttct gctg caca at tt gaac c caa ggac tcca caac gcag acat gcc-g ac tg gctg ctgctggagc tgccaggtgc atggtggagg tgcaagtatc gggaat ccag agcccacatt cccgagcccg ctccaccctt agagagacca ac tgc ct ttg cttggtcttt tttcttagtg utatcatccc aagaacagga tcctctcaag cacgattcag tgagacaacc aagggccgtc cagaggctct aatccagaaa gcagctaaaa tgaaaccctt gagtctgtgg gcccatcatt aattaaatat gtcttaaagt tcattttcac attttataat tatcigcctca ctggtgctgt tcccctggac acactatgag tatcaatcaa.

tgagcaaggc tgtgtttgta gagcagaaga ccttttctac ccttcccgga ctgacttcag aaatgactga ttctggttcc gctggtgctg gaagaagaag aaacaacs gtaggccaca 100 150 200 250 300 350 400 450 500 550 600 650 700 750 aggcat ctgc cttctaqgggg gatggcatga cctcttggga tgtgtgtaat tcttggggtg tggcatgact tcttgggatg tgtgtaatag ttgtgttaag gt taaataaa aaataaagaa atgagtgact tgggtatgaa ctagcacaga tgatatcatc agaaccttct ggtatgaaga agcacagagc atatcatcca aaccttctta ttaaatcatt ctttgtgtat agagtaaact ttaagactca aatgfcttcagr gctgatctct cagtctttat tagcattaag tgcttcagag tgatctctgt gtctttatat gcat taagac tttgtcctaa ttatataata g 1321 aagaccaaac agctcatcscg gtttctgttt atgttgccaa accttgtaaa ctcatgcgcg ttctgttttg gttgccaata cttgtaaaca ttgtaatgtg ataaagctaa actgagcttt cqttacccac tgctttattc tatacctcat caaaaataat ttacccacga ctttattccc tacctcattg aaaataattc taatcttaaa aactgatata 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 too* .5000 0 as 0 Sa *0* <210> <211> 134 <212> PRT <213> Homno sapiens <400> Met 1 Arg Gly Thr Gin Ser Ile Ala Leu Glu Gin Asn Pro Glu Met Thr Leu Gin Leu Pro Glu Pro Val Arg Thr Ser Thr Ala Ser Ser Lys Pro Thr Gly Cys Lys Lys Leu Arg 110 Val Ala Val Ile Arg Gly Asp Pro Leu Tyr Cys Giu Giu Gin Lys Ile Pro Phe Leu Phe Glu Ser Val Ala Asp Gin Pro Ile Thr 25 Ile 40 Lys 55 Met 70 Tyr 85 Phe 100 Ile 115 Cys Lys Tyr Pro Tyr Leu Gly Ile Val Gly Giu Gin Asp Leu Tyr Gly Arg Ala Lys Thr Pro Asp Trp Phe Leu Thr Ser Giu Giu Gin Pro Gin Gly Ile 105 Leu 120 Gly Asp Ala Asp Gly Gly Gly Arg Ala Val Gly Lys Ser Tyr Asn Thr Ala Phe Giu Leu Asn Ile Asn Asp <210> 11 <211> 249 <212> DNA <213> Homno sapiens <400> 11 aagctgctgg agccacga tcttgccagg tgctgaga ctgatggtgg aggaaggg acatgcaagt atccagag tttgggaatc cagaatcc <210> 12 <211> 468 <212> DNA <213> Hoamo sapiens <400> 12 atggtcctga gtggggcg ggtgctttat ctgcataa ggaaggtcat taaaggtg gatgccagcc tgtccccc cctgtcatgt ggggtggg acatcatgga gctctatc taccggcggg acatgggg gggctggttc ctgtgcac cccagcttcc cgagaatg ttccagcagt gtgactag <210> 13 <211> 155 <212> PRT <213> Homno sapiens <400> 13 Met Val Leu Ser Gly 1 5 Leu Lys Val Leu Tyr Ltt cagtccccta Lca accacactat rcc gtctatcaat rgc tcttgagcaa ag aaatgtgttt ct gtgcttccga at ta accagcttct ag aa gagatcagcg tg gt catcctgggt gt gc aggagccgac tc tt ggtgccaagg aa ct cacctccagc tt gg tgcctgaagc cg gt ggctggaatg cc 468 Ala Leu Cys Phe Leu His Asn Asn Val Ile Lys Gly Asp Ala Ser Leu Gin Cys Leu Ser gaag ctgg.

gtcc ccag taac tcca cgagi atcac cccal Arg 10 Gin 25 Glu 40 Ser 55 Cys gact cggcattgaa aggg ctgcatgcag ccaa tcggtggctg ggtg gaagccagtg acta gagccagtga agag cttcaccttc :cgg ctgcctaccc jcct gtcagactca :cac agacttctac Met Lys Asp Ser Leu Leu Ala Gly Giu Ile Ser Val Pro Val Ile Leu Gly Val Gly Gin 100 150 200 250 300 350 400 450 Ala C ly Val Gly Giu gactataaat aaagaccctt gagaggoact ccaggagacg 100 caatcactgt tgctgttatc 150 ggcagagggg atcccattta 200 gtattgtgag aaggttgga 249 Leu His Ala Gly Pro Asn Arg Trp Val Gin Gly Gly Pro Thr Leu Thr Lys Leu Ser Leu Giu Pro Val Asn Ile 85 Met Giu Leu Tyr Leu Gly Ala Lys Giu Gly Leu Thr Ser Leu Cys Thr Val Leu Pro Giu Asn Phe Gin Gin Cys Ser Ser 110 Pro 125 G iy 140 Asp 155 Lys Ser Phe Thr Phe Glu Ser Ala Giu Ala Asp Gin Gly Trp Asn Ala Phe 100 Ala 115 Pro 130 Pro 145 Tyr Arg Arg Asp Tyr Pro Gly Trp Val Arg Leu Thr Ile Thr Asp Phe A <210> 14 <2i1> 295 <212> DNA <213> Homo sapiens <220> <22i> unsure <222> 283 <223> unknown base <400> 14 gctcccgcca ggagaac gctcaagatg gtcctg cattgaaggt gctttal catgcaggga aggtcal gtggctggat gccagcc gccagtgcct gtcatgl <210> <211> 1385 <212> DNA <213> Mus inusculus <400> atagggaatt tggccct agga agtg tctg :taa ctgt :ggg acattctgag gggcgctgtg cataataacc aggtgaagag cccccgtcat gtggggcagg ggccaagaat tttccagcct ggtctacata ccgaatgaag tgctggctgg agtgttgtcc gggcgttcaa caattctgaa gggagtctac cttccgaatg agcttctagc at cagcgtgg cctgggtgtc agncgactct tcggcacgag tgtctttgcc ctgtggagct gattcagcct aggactgcac caaat cgggc ggaggaagcc act tgagc ca accctgtgga aaggactcgg 100 tggagggctg 150 tccccaatcg 200 cagggtggaa 250 aacat 295 cga 1aat tgctgtttat ctgagtgggg gtatctgcac tcattaa-agg agtctgtccc t tgtgggaca t tcaaaatag cactatgctt aataaccagc tgaggagatc ctgtcatcct gagaaagggc gggagcctgc taaaatttcc catgatggtt tgaaggtact gcagagaagg actggatgcc agtgcctatc gtgaacatca tggagctcta cctcgggg cgggatatgg gtcttacc gttcctctgc acctcacc tccctgagga ccccgcct cagtgtgact agggctgc gtaggcagtg gcggctcc agtaggtggc ttactcct aaggcacaca gacactct tggtatttgg agctcaat tctggtgtgg aacccaat aaaagattct tgggtgaa tctgacacag tacctcag tggagggggg gtcaccaa aacctttctg acatctgc cctgaaccga gagggtga cactgtcctg gtttgaaa tctctactca cataaaaa ctactaaata acatacet tatattatat attttaaa aaaaacatgc ggccgcaa <210> 16 <211> 155 <212> PRT <213> Mus muscuius <400> 16 Met Val Lau Ser Gly 1 5 Leu Lys Val Leu Tyr Leu His Ala Glu Lys Prc Asn Arg Ala Lau Val Gin Gly Gly Ser cc aaggaatcaa ag to cagcttcgaa tc gg aagctgacca gc gg gatgctccca to gt ggtccccaaa ac tg atagaggata ga ct ccttccotac tg ct tctcctgcat cc ag aaaccacgta gg ga agaggtggga ac ia gtcctgccat tc ja ctttctctgg ct ig cctctctcat to :a tcaggatagc tg 3c agaggggaca at ;a agcttgtgaa ca' ~a aaaaaaaaaa aa Ala Leu Cys Phe Leu His Asn Asn Val Ilie Lys Gly Asp 'ah Ser e Gin Cys Leu Ser ragcttcac cgctgcct :ctgtcagg acagactt tccataag gagacaga gactcog cagtgctg agattgga accaacaa tgttoata cttatgtt ggctgggc ttgccttc acagaaga aaaaaacc tt aagt gg aattaaaa aaaaaaaa gga 1385 cttctaccgg acccaggctg ctcactcaga ctactittoag cagaggcaga ggagctccac cttctgacct gtaaatcttc tggtactacc agagcaacat catagtaaga ctggagaaag cctttccctc attctctggc tgaccaggca ctgattctgg gaagagattg tatacttctc aaaaaaaaaa 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200 1250 1300 1350 Ala Gly Val Gly Gly Arg 10 Gin 25 Glu 40 55 Cys 70 Met Lys Asp Ser Leu Leu Ala Gly Giu Ile Ser Val Pro Val lie Leu Gly Thr Giu Lys Pro Ile Leu Lys Gly Ala Lys Glu Gly Leu Thr Ser Leu Cys Thr Ser Ile Pro Giu Asp Phe Gin Gin Cys Leu Ser Ser 110 Pro 125 Pro 140 Asp 155 Glu Pro Val Asn Lys Ser Phe Thr Phe Glu Ser Ala Glu Ala Asp Gin Ala Trp Asp Ala Ile 85 Phe 100 Ala 115 Pro 130 Pro 145 Met Glu Leu Tyr Tyr Arg Arg Asp Tyr Pro Gly Trp Val Arg Leu Thr Ile Thr Asp Phe Leu Met 105 Phe 120 Gin 135 Tyr 150 <210> 17 <211> 382 <212> DNA <213> Mus musculus <400> 17 ggagcctgct ttctac' aaaatttcct gctgttl atgatggttc tgagtg gaaggtactg tatctg( cagagaaggt cat taa~ ctggatgcca gtctgt( gtgcctatct tgtggg.

tgaacatcat ggagctc <210> 18 <211> 626 <212> DNA <213> Homo sapiens <400> 18 aaaatgggct ctgaggz agacccggct gtaagcc attttgttca cacaagt agcattcatg accaggz catagcagtt ccagate tagcctcatc cttgagc ctgggggtct ctaaagg acaaagtcat ccatccc t tag tat t 999C caca 3.ggt cccc Fcag ftac gtctcaaatt tcaaaatagg actatgcttc ataaccagct gaggagat ca tgtcatcctg agaaagggcc ctcggggcca ggaaaaagat tggaaccagg aaggtgaaga caaagtactg actacatacg gcctctgcgg gttttgtCtc agctgaagaa ttccagcctt gtctacatac cgaatgaagg gctggctgga gtgttgtccc ggcgttcaag aattctgaaa ag 382 gaaccccagt cccaagcctc acttaaaccc gtcctggact cccagagatc agaaaggaag tactgtgaca ggagaaactg gtctttgcct tgtggagctc 100 attcagcctt 150 ggactgcacg 200 aaatcgggca 250 gaggaagcca 300 cttgagccag 350 gctgcttaga cccgccatga 100 gaagaaattc 150 ctgggaatct 200 ttctttgcat 250 tccgattctc 300 aggataaagg 350 atgaagctgg 400 ~ctg ccc :cca ~tca ~aaa t ca rgga :ttc ctgcccaaaa ggaatcagca cgccggccct gtgggctcct ggaacatgct ggagtcggcg ctgcacctcc tgcaattgta atgagcctgt agaacaggaa acacattgaa ttttcatttc atgagcccca gtgaggtcag ogatta 626 <210> 19 <211> 207 <212> PRT <213> Homo sapiens tcatctttta tagggctcag 450 gctcaccccg gatggttcat 500 tggggtgaca gataaatccg 550 aaccagtttg caaagctgaa 600 <400> 19 Met Gly Ser Giu Asp 1 Glu Ala Pro Leu Arg Ser Glu Ser Lys Gly Ile Lys Asp Pro Met Asn Lys Lys Asp Ser Pro Glu Ala Glu Phe Cys Leu Gin Glu Ser Ser Trp Cys Thr Phe Glu 5 Ala Val Phe Val Phe Ser Gly Asn Ile Phe Lys Gly Leu Tyr 110 Leu Lys 125 Ala Arg 140 Asn Met 155 Ser Cys 170 Asn Arg 185 Trp Ser His Ile Leu Phe Ser Cys Lys Arg Leu Asn Lys Glu Pro Thr His Ile Ala Pro Asp Glu Pro Glu Cys His Lys Leu Ser Asp Ala Leu Ile Lys Lys Phe Ser Asn Ile Asp Glu Pro Gin Va1 Ala Leu Asp Leu Ile Ala Glu Glu Glu 10 Pro 25 Lys 40 Asp 55 Pro 70 Ser 85 Leu 100 Lys 115 Met 130 Phe 145 Ala 160 Pro 175 Phe 190 Pro Gly Val His Asp Ser Gly Gly Lys Tyr His Val Ser Gln Pro Lys Lys Lys Leu Va1 Gin Leu Arg Pro Gly Phe Cy Ser Asn Va1 Asn Ser Ser Ser Ala Ala Gly Va1 Gin Cys Leu Leu Leu Tyr Ser Lys His Ala Gin Trp Thr Pro Leu Pro Asn Vai Ile Ala Gly 105 Pro 120 Gin 135 Val 150 Phe 165 Asp 180 Val 195 Cys Lys Ala Giu Met Ser Pro Ser Giu Val Ser Asp 200 205 <210> <211> 506 <212> DNA <213> Homo sapiens <400> aaaatgggct ctgaggactg agacccggct gtaagccccc attttgttca cacaaagatc gcctctgcgg agaaaggaag gttttgtctc tactgtgaca agctgaagaa ggagaaactg cgccggccct tcatctttta ggagtcggcg gctcaccccg atgagcctgt tggggtgaca ttttcatttc aaccagtttg cgatta 506 <210> 21 <211> 167 <212> PRT <213> Homo sapiens ggaaaaagat tggaaccagg ttctttgcat tccgattctc aggataaagg atgaagctgg tagggctcag gatggttcat gataaatttg caaagctgaa gaaccccagt cccaagcctc tagcctcatc ctgggggtct acaaagtcat ctgcccaaaa gtgggctcct ctgcacctcc agaacaggaa atgagcccca gctgcttaga cccgccatga cttgagctca ctaaagggga ccatcccttc ggaat cagca ggaacatgct tgcaattgta acacattgaa gtgaggtcag 100 150 200 250 300 350 400 450 500 a a <400> 21 a.

1 Glu Gly Ser Glu Asp Pro Ala Ala Met Asn Phe Ser Leu Ser Ser Gly Val Ser Lys Gly Gin Ser His Lys Leu Ala Ala Tyr Arg Ala Gin His Pro Gly Trp Val Gly Val Thr Asp 5 Val1 Val Ala Gly Pro Gin Val 110 Phe 125 Asp 140 Ser Pro Leu Glu His Thr Lys Ile Ser Ala Glu Lys Glu Phe Cys Leu Ser Leu Gin Leu Lys Glu Ser Ala Gly Ser Trp Asn Ile Cys Thr Ser Pro 25 Phe 40 Giy 55 Tyr 70 Lys 85 Arg 100 Met 115 Cys 130 Gly Pro Ser Leu Phe Ala Leu Ala Ser Pro Ile Leu Cys Asp Lys Asp Lys Giu Lys Leu Arg Pro Phe Ile Leu Glu Ser Ala Asn Cys Asn Giu Trp Giu Lys Asp Glu Pro Gin Cys Cys Leu Pro Ser Leu Lys Met Phe 105 Ala 120 Pro 135 Lys Phe Giu Asn Arg Lys His Ile Glu Phe 14 Ser Phe Gin Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val 155 160 165 Ser Asp <210> 22 <211> 561 <212> DNA <213> Homno sapiens <400> 22 aacccgaaga aattcagcat tcatgaccag gatcacaaag tactggtcct ggactctggg aatctc agatcttctt tgcatt ggaagtccga ttctcc tgacaaggat aaagga aactgatgaa gctggc ttttataggg ctcagg ccccggatgg ttcatc tgacagataa atttga gtttgcaaag ctgaaa cccattgaac gccttc aacctgctca c 561 <210> 23 <211> 561 <212> DNA <213> Homno sapiens <400> 23 ttgggcttct ttaagt cctgagaccc ttagagl tctagaagaa acgtaal ccttcaggct aagagg actgttccta tttcctc ttgactiactt cgaccg aaaatatccc gacrtcc atag agc c tggg caaa tgcc t ggg tgca gaac tgag ctcg cagttccaga tcatccttga ggtctctaaa gtcatccatc caaaaggaat ctcctggaac cctcctgcaa aggaaacaca ccccagtgag ctaatttgaa agtactggtc gtcaaggtct agtaggaact ccagagattt cagtaggtag gttttcctta gaggacct tg ggaggacgt t tcctttgtgt taaaaactac gctcagcctc ggggagtttt ccttcagctg cagcacgccg atgctggagt t tgtaatgag ttgaattttc gtcagcgatt ctaattgtat ctagtgtttc atttttgatg cgagtcggag cccctcaaaa ggaagt egac gtcgtgcggc tacgacc'tca aacattactc aacttaaaag atacgcccag 100 tgcggagaaa 150 gtctctactg 200 aagaaggaga 250 gcccttcatc 300 cggcggctca 350 cctgttgggg 400 atttcaacca 450 aggaaactgc 500 aaaaacacca 550 atgaccagga tatgcgggtc 100 acgcctcttt 150 cagagatgac 200 ttcttcctct 250 cgggaagtag 300 gWccgWccgagt 350 ggacaacccc 400 taaagttggt 450 cgta :atc :cgg ccC t t t Icgg

~CCC

~cgt Ztg ggggcctacc actgtctatt aagtag taaactc caaacgtttc gacttactc ggggtcactc cagtcgctaa tcctttgacg 500 gggtaacttg cggaaggagc gattaaactt gattaacata tttttgtggt 550 ttggacgagt g 561 <210> 24 <211> 839 <212> DNA <213> Homo sapiens 00 <400> 24 ggccctcgag agtaataaac tgaaaatggg gaagacccgg gaattttgtt tcagcattca ctcatagcag attagcctca tcctgggggt ggacaaagtc ggctgcccaa aggtgggctc atctgcacct tgagaacagg aaatgagccc ttcctcgcta aaaaaaaaaa <210> <211> 218 <212> PRT gccaagaat t tcaacgttga ctctgaggac ctiggaagccc cacacaagt c tgaccaggat ttccagataa tccttgagct ctctaaaggg atccatccct aaggaat cag ctggaacatg cctgcaattg aaacacattg cagtgaggtc atttgaacta cggcacgagg aaatgtcctt t gggaaaaag cctggaacca caaaggtgaa cacaaagtac aaactacata cagcctctgc gagttttgtc tcagctgaag cacgccggcc ctggagtcgg taatgagcct aattttcatt agcgattagg attgtataaa cttcattcca tgtgggggag atgaacccca ggcccaagcc gaacttaaac tggtcctgga cgcccagaga ggagaaagga t ctao tgt ga aaggagaaac cttcatcttt cggctcaccc gt tggggtga tcaaccagtt aaactgcccc ttttctgttg aactcaggag 100 gtgctgctta 150 tcccaccat 200 ccgaagaaat 250 ctctgggaat 300 tcttctttgc 350 agtccgattc 400 caaggataaa 450 tgatgaagct 500 tatagggctc 550 cggatggttc 600 cagataaatt 650 tgcaaagctg 700 attgaacgcc 750 aacaccaaac ctgctcacta 800 aaaaaaacgt ttgcggccgc aagcttatt 839 <213> Homo sapiens <400> Met Ser Phe Val Gly Glu Asn Ser Gly Val Lys Met Gly Ser Clu 1 5 10 Asp Trp Glu Lys Asp Glu Pro Gin Cvs Cys Leu 0111 Asp Pro Ala 25 Gly Ser Pro Leu Glu Pro Gly Pro Ser Leu Pro Thr Met Asn Phe 40 16 Val His Thr Ser Pro Lys Val Lys Asn Leu Asn Pro Lys Lys Phe 55 Ser Ile His Asp Gin Asp His Lys Val Leu Val Leu Asp Ser Gly 70 Asn Leu Ile Ala Val Pro Asp Lys Asn Tyr Ile Arg Pro Giu Ile 85 Phe Phe Ala Leu Ala Ser Ser Leu Ser Ser Ala Ser Ala Giu Lys 100 105 Gly Ser Pro Ile Leu Leu Gly Val Ser Lys Gly Giu Phe Cys Leu 110 115 120 Tyr Cys Asp Lys Asp Lys Gly Gin Ser His Pro Ser Leu Gin Leu 125 130 135 Lys Lys Giu Lys Leu Met Lys Leu Ala Ala Gin Lys Giu Ser Ala 140 145 150 Arg Arg Pro Phe Ile Phe Tyr Arg Ala Gin Val Giy Ser Trp Asn *155 160 165 Met Leu Giu Ser Ala Ala His Pro Gly Trp Phe Ile Cys Thr Ser 170 175 180 Cys Asn Cys Asn Giu Pro Val Gly Val Thr Asp Lys Phe Giu Asn :185 190 195 Arg Lys His Ile Giu Phe Ser Phe Gin Pro Val Cys Lys Ala Giu 200 205 210 Met Ser Pro Ser Giu Val Ser Asp ****215 <210> 26 <211> 177 <212>

PRT

0 00<213> Homo sapiens 0 00 :0 <400> 26 Met Giu Ile Cys Arg Gly Leu Arg Ser His Leu Ile Thr Leu Leu 1 5 10 Leu Phe Leu Phe His Ser Giu Thr Ile Cys Arg Pro Ser Gly Arg 25 Lys Ser Ser Lys Met Gin Ala Phe Arg Ile Trp Asp Val Asn Gin 40 Lys Thr Phe Tyr Leu Arg Asn Asn Gin Leu Vai Ala Gly Tyr Leu 55 Gin Gly Pro Asn Val Asn Leu Giu Giu Lys Ile Asp Val Vai Pro 70 Ile Glu Pro His Ala Leu Phe Leu Gly Ile His Gly Gly Lys Met 85 Cys Leu Ser Cys Val Lys Ser Gly Asp Giu Thr Arg Leu Gin Leu

S

S

Glu Ala Val Asn Ile 110 Lys Arg Phe Ala Phe 125 Phe Glu Ser Ala Ala 140 Glu Ala Asp Gin Pro 155 Val Met Val Thr Lys 170 <210> 27 <211> 169 <212> PRT <213> Homo sapiens <400> 27 Met Arg Gly Thr Pro 1 5 Tyr Gin Ser Met Cys Asn Gin Gin Val Trp Pro Arg Ser Asp Ser 50 Cys Lys Tyr Pro Glu Tyr Leu Gly Ile Gin Val Gly Glu Gin Pro Asp Leu Tyr Gly Gin 110 Arg Ala Lys Thr Gly 125 Pro Asp Trp Phe Ile 140 Leu Thr Ser Glu Leu 155 Asn Ile Asn Asp <210> 28 <211> 167 Thr Iie Cys Val Phe Gly Lys Thr Val Ala Asn Thr Pro Arg Ala Asp Arg Pro Ser Tyr Asp Pro Leu Thr Leu Pro Leu Glu Thr Ser Leu Ser Gly Leu Phe Ala Ile Gin Pro Glu Glu Gin Pro Ser Ser Ser Asp Trp Thr Gin Asp Thr Gly Val Gin Met Leu Val Thr Lys 17 100 Glu 115 Ser 130 Phe 145 Asn 160 Glu 175 Gly 10 Gly 25 Gin 40 Thr 55 Gly 70 Cys 85 Lys 100 Lys 115 Leu 130 Arg 145 Asn Gly Leu Met Asp Gly Thr Asn Val Arg Leu Glu Pro Glu Asp Arg Pro Cys Pro Glu Gly Ile Leu Ala Gly Tyr Gin Phe Ser Gin Lys Gin Thr Thr Thr Ala Asp Glu Arg Asn Val Val Asp Cys Lys Leu Val Pro Ala Asp Ala Ile Pro Glu Ile Phe Ala Ile 105 Asp 120 Ser 135 Met 150 Gly 165 Val Leu Val Thr Ile Lys Met 105 Tyr 120 Phe 135 Ile 150 Gly Lys Ser Tyr Asn Thr Ala Phe Glu Leu 160 165 <212> PRT <213> Homo sapiens <400> 28 Met Gly Ser Giu Asp Trp Giu Lys Asp Glu Pro Gin Cys Cys Leu 1 Glu Thr Ser Gly G ly Lys Tyr His Val1 Asp Met Leu Val1 Gin Leu Arg Pro G ly Pro As n Ser Ser Ser Ala Ala Gly Val Ala Phe Ser Ly s His Ala Gin Trp Thr 5 Gly Val1 Ala Gly Pro 80 Gin Val1 110 Phe 125 Asp 140 10 Ser His Ser Glu Ser Lys Gly Ile Lys Pro Thr Ala Phe Leu Glu Ser Cys Phe Leu Ly s Giu Cy s Gin Ser Trp Thr Glu Glu Ile Lys Leu Leu Ala Asn Ser Asn Pro 25 Phe 40 Gly 55 Tyr 70 Lys 85 Arg 100 Met 115 Cys 130 Arg 145 Gly Phe Ser Cy s Lys Arg Leu Asn Lys Pro Ala Pro Asp Glu Pro Giu Cys His Ser Leu Ile Lys Lys Phe Ser Asn Ile Leu Ala Leu Asp Leu Ile Ala Giu Glu Pro Ser Leu Lys Met Phe 105 Ala 120 Pro 135 Phe 150

S

S

SS 55 S S 5 5 555 5 55

S

5555

S

55 S S

SSSSSS

S

Ser Phe Gin Pro Val Cys Lys Ala Glu Met Ser Pro Ser Glu Val 155 160 165 Ser Asp <210> 29 <211> 31 <212> DNA <213> Homo sapiens <400> 29 ggcggatcca aaatgggctc tgaggactgg g 31 <210> <211> <212> DNA <213> Homo sapiens <400> gcggaattct aatcgctgac ctcactgggg <210> 31 <211> 9 <212> PRT <213> Homo sapiens 19 <400> 31 Val His Thr Ser Pro Lys Val Lys Asn 1 <210> 32 <211.> <212> PRT <213> Homno sapiens <400> 32 Val Leu Ser Gly Ala Leu Cys Phe Arg Met 1 5 0 S

Claims (63)

1. An isolated IL-llp polypeptide that binds to an IL-18R ECD, but not to an IL- 1R ECD, the polypeptide having: an hIL-1Ral amino acid sequence of residues 37 to 203 of Figure 2 (SEQ ID NO:5), or variants thereof having from 1 to 5 additions, deletions or conservative substitutions, or an hIL-1Ral amino acid sequence of residues 15 to 193 of Figure 3 (SEQ ID NO:7) or variants having from 1 to 5 additions, deletions or conservative substitutions.

2. The IL-llp polypeptide of Claim 1, wherein the variant has from additionally substituted amino acid residues.

3. The IL-llp polypeptide of Claim 1, wherein the variant has from 1-5 deleted amino acid residues

4. The IL-llp polypeptide of Claim 1, wherein the variant has from conservatively substituted residues. The IL-llp polypeptide of Claim 1, consisting of the sequence of residues from about 37 to about 203 of Figure 2 (SEQ ID

6. The IL-llp polypeptide of Claim 1, consisting of the sequence of residues from about 15 to about 193 of Figure 3 (SEQ ID NO:7).

7. An isolated IL-llp polypeptide that binds to an IL-1R ECD, but not to an IL- 18R ECD, the polypeptide being an hIL-1RalS polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence of residues 26 to 167 of Figure 16 (SEQ ID NO:21). 25 8. The IL-llp polypeptide of Claim 7, wherein the sequence identity is

9. The IL-p polypeptide of Claim 7, wherein the sequence identity is The IL-llp polypeptide of Claim 7, wherein the sequence identity is The IL-llp polypeptide of Claim 7, wherein the sequence identity is

11. The IL-11p polypeptide of Claim 7, which comprises a sequence of residues 26 to 167 of Figure 16 (SEQ ID NO:21). 30 12. A chimeric polypeptide comprising the IL-llp polypeptide of Claim 6 or Claim 7, fused to a heterologous amino acid sequence. Se 13. The chimeric polypeptide Claim 12, wherein said heterologous sequence is an epitope tag sequence.

14. The chimeric polypeptide of Claim 12, wherein said heterologous sequence is an Fc region of an immunoglobulin. An isolated IL-llp polypeptide consisting of the sequence of residues from 37 to 203 of Figure 2 (SEQ ID

16. An isolated IL-llp polypeptide consisting of the sequence of residues from to 193 of Figure 3 (SEQ ID NO:7).

17. An isolated polypeptide comprising SEQ ID NO:2. -83- H:\Juanita\Kecp\patent\25935.4.doc 8/10/04

18. An IL-lip polypeptide consisting of an amino acid sequence selected from the group consisting of: a 26 amino acid IL-11p N-terminal polypeptide encoded by the cDNA insert in the vector deposited as ATCC Dep. No. 203588; and a polypeptide encoded by the cDNA insert in the vector deposited as ATCC Dep. No. 203587, the polypeptide binding to an IL-18R ECD but not to an IL-1R ECD, including or alternatively excluding a 36 N-terminal amino acid residue sequence.

19. An IL-llp polypeptide comprising an amino acid sequence that binds to an IL- 1R ECD but not to an IL-18R ECD, encoded by the cDNA insert in the vector deposited as ATCC Dep. No. 203855. An isolated DNA molecule encoding an IL-11p polypeptide that binds to an IL- 18R ECD, but not to an IL-1R ECD, selected from the group consisting of: an hIL-Ral amino acid sequence of residues 37 to 203 of Figure 2 (SEQ ID NO:5) or variants thereof having from 1-5 additions or deletions or conservative substitutions, an hIL-1Ral amino acid sequence of residues 15 to 193 of Figure 3 (SEQ ID NO:7) or variants thereof having from 1-5 additions, deletions or conservative substitutions, and the complement of the DNA molecules of

21. An isolated DNA molecule encoding an IL-llp polypeptide that binds to an IL- 18R ECD, but not to an IL-1R ECD, selected from the group consisting of: an hIL-Ral amino acid sequence of residues 1 to 203 of Figure 2 (SEQ ID NO:5) or variants thereof having from 1-5 additions, deletions or 25 conservative substitutions, an hIL-1Ral amino acid sequence of residues 1 to 193 of Figure 3 (SEQ ID NO:7) or variants thereof having from 1-5 additions, deletions or conservative substitutions, and the complement of the DNA molecules of 30 22. The DNA molecule of Claim 20 or Claim 21, wherein the variant has from additionally substituted amino acid residues. •23. The DNA molecule of Claim 20 or Claim 21, wherein the variant has from deleted amino acid residues.

24. The DNA molecule of Claim 20 or Claim 21, wherein the variant has from conservatively substituted residues. The DNA molecule of Claim 20, wherein the encoded IL-llp polypeptide is a sequence of residues from 37 to 203 of Figure 2 (SEQ ID

26. The DNA molecule of Claim 20, wherein the encoded IL-llp polypeptide is a sequence of residues from 15 to 193 of Figure 3 (SEQ ID NO:7). -84- H:\Juanita\Keep\patent\25935.4.doc 8/ 10/04

27. The DNA molecule of Claim 21, wherein the encoded IL-11p polypeptide is a sequence of residues from 1 to 203 of Figure 3 (SEQ ID

28. The DNA molecule of Claim 21, wherein the encoded IL-llp polypeptide is a sequence of residues from 1 to 193 of Figure 3 (SEQ ID NO:7).

29. An isolated DNA molecule encoding an IL-llp polypeptide that binds to an IL- 1R ECD, but not to an IL-18R ECD, the DNA molecule encoding an hIL-1RalS polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence of residues 26 to 167 of Figure 16 (SEQ ID NO:21) or the complement of said DNA molecule. An isolated DNA molecule encoding an IL-llp polypeptide that binds to an IL- 1R ECD, but not to an IL-18R ECD, the DNA molecule encoding an hIL-1RalS polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence of residues 1 to 167 of Figure 16 (SEQ ID NO:21) or the complement of said DNA molecule.

31. The isolated DNA molecule of Claim 29 or Claim 30, wherein the sequence identity is

32. The isolated DNA molecule of Claim 29 or Claim 30, wherein the sequence identity is

33. The isolated DNA molecule of Claim 29 or Claim 30, wherein the sequence identity is

34. The isolated DNA molecule of Claim 29, wherein the encoded IL-llp polypeptide comprises residues 26 to 167 of Figure 16 (SEQ ID NO:21). The isolated DNA molecule of Claim 30, wherein the encoded IL-llp polypeptide comprises res residues 1 to 167 of Figure 16 (SEQ ID NO:21). *36. An isolated DNA molecule encoding the polypeptide of any one of Claims 15 to 17. 25 37. A vector comprising the DNA molecule of Claim

38. A vector comprising the DNA molecule of Claim 26.

39. A vector comprising the DNA molecule of Claim 27. A vector comprising the DNA molecule of Claim 28.

41. A vector comprising the DNA molecule of Claim 34.

42. A vector comprising the DNA molecule of Claim

43. A vector comprising the DNA molecule of Claim 36. o* 44. A vector according to any one of Claims 37 to 43 operably linked to control :0 sequences recognized by a host cell transfected with the vector.

45. A host cell comprising the vector according to any one of Claims 37 to 44.

46. A process for producing an IL-llp polypeptide, comprising the steps of: culturing a host cell comprising the DNA molecule of any one of Claims 25 to 28 or 34 to 36 under conditions suitable for the expression of the IL-llp polypeptide encoded by the DNA molecule; and recovering said IL-llp polypeptide from the cell culture. H:\Juanita\Keep\palent\25935.4.doc 8/10/04

47. An antagonist of IL-llp activity that partially or fully blocks, inhibits or neutralizes binding of a polypeptide of any one of claims 1 to 6 an IL-18R ECD, which antagonist comprises an antagonist antibody or antibody fragment, specific for a polypeptide according to any one of Claims 1 to 6.

48. An antagonist of IL-llp activity that partially or fully blocks, inhibits or neutralises binding of a polypeptide of any one of Claims 7 to 11 an IL-1R ECD, which antagonist comprises an antagonist antibody or antibody fragment, specific for a polypeptide according to Claims 7 to 11.

49. An antagonist according to Claim 47 or Claim 48, wherein the antibody is a monoclonal antibody. An antagonist according to Claim 47 or Claim 48, wherein the antibody is a human antibody.

51. An antagonist according to Claim 47 or Claim 48, wherein the antibody is a humanised antibody.

52. A pharmaceutical composition comprising an IL-llp polypeptide according to any one of Claims 1 to 19, or an antagonist according to any one of Claims 47 to 51, together with a pharmaceutically acceptable carrier.

53. A method of treating an inflammatory disorder disease, comprising administering to a mammal in need thereof an effective amount of a pharmaceutical composition according to Claim 52.

54. Use of an IL-11p polypeptide according to any one of Claims 1 to 19, or an antagonist according to any one of Claims 47 to 51, in the manufacture of a medicament for use in treating an inflammatory disorder or disease.

55. A method according to Claim 53 or use according to Claim 54, wherein the 25 inflammatory disorder or disease includes inflammatory skin diseases such as psoriasis and atopic dermatitis; systemic scleroderma and sclerosis; responses associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); ischemic reperfusion disorders including surgical tissue reperfusion injury, myocardial ischemic f*ew, conditions such as myocardial infarction, cardiac arrest, reperfusion after cardiac surgery 0. 30 and constriction after percutaneous transluminal coronary angioplasty, stroke, and abdominal aortic aneurysms; cerebral edema secondary to stroke; cranial trauma; hypovolemic shock; asphyxia; adult respiratory distress syndrome; acute lung injury; *0 Behcet's Disease; dermatomyositis; polymyositis; multiple sclerosis; dermatitis; meningitis; encephalitis; uveitis; osteoarthritis; autoimmune diseases such as rheumatoid arthritis, Sjorgen's syndrome, vasculitis, and insulin-dependent diabetes mellitus (IDDM); diseases involving leukocyte diapedesis; central nervous system (CNS) inflammatory disorder; meningitis; multiple organ injury syndrome secondary to septicaemia or trauma; inflammatory diseases of the liver, including alcoholic hepatitis and hepatic fibrosis; pathologic host responses to infection, including pathologic inflammation in granulomatous diseases, hepatitis, and bacterial pneumonia; antigen-antibody complex mediated diseases -86- H:\Juanita\Keep\patent\25935.4.doc 8/10/04 including glomerulonephritis; sepsis; sarcoidosis; immunopathologic responses to tissue/organ transplantation, including graft-versus host disease (GVHD); inflammations of the lung, including pleurisy, alveolitis, vasculitis, pneumonia, chronic bronchitis, bronchiectasis, diffuse panbronchiolitis, hypersensitivity pneumonitis, idiopathic pulmonary fibrosis (IPF), and cystic fibrosis; inflammation in renal diseases, including acute or chronic nephritic conditions such as lupus nephritis; pancreatitis.

56. A method according to Claim 53 or use according to Claim 54, wherein the inflammatory disorder or disease is selected from rheumatoid arthritis, osteoarthritis, sepsis, acute lung injury, adult respiratory distress syndrome, idiopathic pulmonary fibrosis, ischemic reperfusion (including surgical tissue reperfusion injury, stroke, myocardial ischemia, and acute myocardial infarction), asthma, psoriasis, graft-versus-host disease (GVHD), and inflammatory bowel disease such as ulcerative colitis.

57. A method or use according to Claim 56, wherein the inflammatory disorder or disease is allergic asthma.

58. A method of treating an IL-1-mediated disorder, comprising administering to a mammal in need of such treatment an effective amount of an IL-llp polypeptide according to any one of Claims 1 to 11.

59. Use of an IL-llp polypeptide according to any one of Claims 1 to 11 in the manufacture of a medicament for use in treating an IL-1-mediated disorder.

60. A method according to Claim 58 or use according to Claim 59, wherein the IL- llp polypeptide is selected from the group consisting of hIL-1Ral, hIL-1RalL, hIL-1RalV, hIL-1RalS and hIL-1Ra2. S61. A method of treating an IL-18-mediated disorder, comprising administering to a mammal in need of such treatment an effective amount of an IL-llp polypeptide according S: 25 to any one of claims 1 to 11.

62. Use of an IL-llp polypeptide according to any one of claims 1 to 11 in the manufacture of a medicament for use in treating an IL-18-mediated disorder.

63. A method according to Claim 61 or use according to Claim 62, wherein the IL- lip polypeptide is selected from the group consisting of hIL-1Ral, hIL-1RalL, hIL-1RalV, 30 hIL-1RalS and hIL-1Ra2.

64. A method according to Claim 61 or use according to Claim 62 wherein the IL- lip polypeptide is a native sequence hIL-1Ral.

65. A method of treating an inflammatory disorder, comprising administering to a mammal in need of such treatment an effective amount of an IL-llp according to any one of Claims 1 to 19.

66. Use of an IL-llp according to any one of Claims 1 to 19 in the manufacture of a medicament for use in treating an inflammatory disorder.

67. A method according to Claim 65 or use according to Claim 66, wherein the IL- llp is selected from the group consisting of hIL-1Ral and hIL-1RalS. -87- H:\Juanita\Keep\patent\25935.4doc 8/ 10/04

68. A method according to Claim 65 or use according to Claim 66, wherein the IL- 1lp is a native sequence hIL-1Ral.

69. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is asthma.

70. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is rheumatoid arthritis.

71. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is osteoarthritis.

72. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is sepsis.

73. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is acute lung injury.

74. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is adult respiratory distress syndrome.

75. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is idiopathic pulmonary fibrosis.

76. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is ischemic reperfusion disease, such as surgical tissue reperfusion injury, stroke, myocardial ischemia, or acute myocardial infarction.

77. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is psoriasis.

78. A method according to Claim 65 or use according to Claim 66, wherein the inflammatory condition is graft-versus-host disease (GVHD).

79. A method according to Claim 65 or use according to Claim 66, wherein the 25 inflammatory condition is an inflammatory bowel disease such as ulcerative colitis.

80. A IL-llp polypeptide according to any one of Claims 1 to 19, a DNA molecule according to any one of Claims 20 to 36, a vector according to any one of Claims 3 7 to 45, a e* host cell according to Claim 46, an antagonist according to any one of Claims 47 to 51, or a pharmaceutical composition according to Claim 52, substantially as described herein with reference to any one of the examples and/or figures.

81. A method or use according to any one of Claims 46 or 53 to 79, substantially as described herein with reference to any one of the examples and/or figures. Dated this 8 th day of October 2004. GENENTECH. INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia -88- H:\Juanita\Keep\patent\25935.4.doc 8/10/04